Anti-ox40 antibodies and their uses

ABSTRACT

The present disclosure provides novel anti-OX40 antibodies, compositions including the antibodies, nucleic acids encoding the antibodies, and methods of making and using the same.

1. CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 62/434,761, filed Dec. 15, 2016, thecontents of which are incorporated herein in its entirety by referencethereto.

2. SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 30, 2017, isnamed 381493-368US_SL.txt and is 96,788 bytes in size.

3. TECHNICAL FIELD

The present application pertains to, among other things, novel anti-OX40antibodies, compositions including the antibodies, nucleic acidsencoding the antibodies, and methods of making and using the same.

4. BACKGROUND

Cancer therapies comprise a wide range of therapeutic approaches,including surgery, radiation, and chemotherapy. While the variousapproaches allow a broad selection of treatments to be available to themedical practitioner to treat the cancer, existing therapeutics sufferfrom a number of disadvantages, such as a lack of selectivity oftargeting cancer cells over normal, healthy cells, and the developmentof resistance by the cancer to the treatment.

Recent approaches based on targeted therapeutics, which interfere withcellular processes of cancer cells preferentially over normal cells,have led to chemotherapeutic regimens with fewer side effects ascompared to non-targeted therapies such as radiation treatment.

Cancer immunotherapy has emerged as a promising therapeutic approach tocomplement existing standards of care. See, e.g., Miller, et al. CancerCell, 27, 439-449 (2015). Such immunotherapy approaches include thedevelopment of antibodies used to modulate the immune system to killcancer cells.

Anti-tumor immune responses in patients with solid tumors have beenenhanced by treatment with biologics. For example, there are twoapproved and marketed anti-PD-1 monoclonal antibodies: nivolumab(OPDIVO®) and pembrolizumab (KEYTRUDA®), with approvals in the US andthe European Union to treat diseases such as unresectable or metastaticmelanoma and metastatic non-small cell lung cancer. Treatment ofpatients with these agents has resulted in anti-tumor responses asmeasured by improvement in either progression free survival and/oroverall survival.

The recent failure of OPDIVO® to slow progression of advanced lungcancer in a treatment-naïve patient population in a clinical trialcomparing OPDIVO® with conventional chemotherapy highlights the need foralternative approaches and additional cancer treatments to complementexisting therapeutic standards of care.

5. SUMMARY

The present disclosure provides anti-OX40 antibodies that specificallybind to and activate OX40. The amino acid sequences of exemplarycomplementarity determining regions (CDRs), the heavy chain variabledomain (V_(H)) and light chain variable domain (V_(L)) regions (i.e.,the V_(H) and V_(L) chains, respectively), and the heavy and lightchains of exemplary anti-OX40 antibodies are provided in the DetailedDescription below. Anti-OX40 antibodies provided herein result inactivation of the adaptive immune response.

The anti-OX40 antibodies may include modifications and/or mutations thatalter the properties of the antibodies, such as those that increasehalf-life, increase or decrease antigen-dependent cellular cytotoxicity(ADCC), as is known in the art.

Nucleic acids comprising nucleotide sequences encoding the anti-OX40antibodies of the disclosure are provided herein, as are vectorscomprising nucleic acids. Additionally, prokaryotic and eukaryotic hostcells transformed with a vector comprising a nucleotide sequenceencoding a disclosed anti-OX40 antibody are provided herein, as well aseukaryotic (such as mammalian) host cells engineered to express thenucleotide sequences. Methods of producing antibodies, by culturing hostcells and recovering the antibodies are also provided, and discussedfurther in the Detailed Description below.

In another aspect, the present disclosure provides compositionsincluding the anti-OX40 antibodies described herein. The compositionsgenerally comprise one or more anti-OX40 antibodies as described herein,and one or more excipients, carriers or diluents.

The present disclosure provides methods of treating subjects, such ashuman subjects, i.e., human patients, diagnosed with a solid tumor withan anti-OX40 antibody. The method generally involves administering tothe subject an amount of an anti-OX40 antibody described hereineffective to provide therapeutic benefit. The subject may be diagnosedwith any one of a number of solid tumors that may be newly diagnosed,relapsed, or relapsed and refractory. An anti-OX40 antibody can beadministered as an intravenous infusion once every two weeks.

The anti-OX40 antibodies may be administered as single therapeuticagents (monotherapy) or adjunctive to or with other therapeutic agentstypically, but not necessarily, those used for the treatment of a solidtumor. Therapeutic agents typically will be used at their approved dose,route of administration, and frequency of administration.

The anti-OX40 antibodies may be administered via a variety of routes ormodes of administration, including but not limited to, intravenousinfusion and/or injection, and intratumoral injection. The amountadministered will depend upon the route of administration, the dosingschedule, the type of cancer being treated, the stage of the cancerbeing treated, and other parameters such as the age and weight of thepatient, as is well known in the art.

6. BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D depict functional activation of human T cells in vitro aftertreatment with the exemplary anti-OX40 antibody Hu3738. FIG. 1A depictsthe proliferation of human peripheral blood CD4+ T cells after treatmentwith anti-OX40 antibody Hu3738, or literature antibody 11D4 or 18D8.FIG. 1B depicts the increase in interferon-gamma (IFN-γ) production byhuman CD4+ T cells after treatment with anti-OX40 antibody Hu3738, orliterature antibody 11D4 or 18D8. FIG. 1C depicts the proliferation ofhuman peripheral blood CD4+ T cells after treatment with Hu3738, orliterature antibody 1A7. FIG. 1D depicts the increase in IFN-γproduction by human CD4+ T cells after treatment with Hu3738, orliterature antibody 1A7.

FIGS. 2A-2B show the effect of exemplary anti-OX40 antibody Hu3738 onhuman T regulatory (Treg) cell-mediated suppression in vitro. The Tregsuppression assay was set up using two different ratios of CD4+/CD25−responder T cells (Tresp) to CD4+/CD25+/CD127low T regulatory cells(Treg). Treg Suppression Inspector reagent beads (Insp) were added toculture wells at 1:1 bead-to-cell ratio for stimulation. The clear barrepresents proliferation of Tresp cells in the presence of Insp.Anti-OX40 and isotype control human IgG₁ antibodies were tested intriplicate at 10 μg/mL final concentration in the absence or presence ofcross-linking reagent (F(ab′)₂ goat anti-human IgG, Fc specific) at 1:4ratio. Plates were incubated at 37° C. in 5% CO₂ for four days. 1³H-thymidine was added and the plates were further incubated for another16 hours. Graphs represent proliferation as shown in counts per minute(cpm). FIG. 2A depicts results with Tresp to Treg at 2:1 ratio; FIG. 2Bdepicts results with Tresp to Treg at 4:1 ratio.

FIG. 3 depicts the inhibition of binding of exemplary anti-OX40 antibodyHu3738 in the presence of soluble human OX40 ligand (OX40L). The graphshows mean fluorescence intensity (MFI) vs. concentration of OX40L(μg/mL). Human OX40-expressing Jurkat cells were co-stained with atitration of unlabeled soluble OX40L and 0.2 μg/mL Hu3738 or isotypecontrol antibody.

FIG. 4A shows an amino acid sequence alignment of human OX40 (SEQ IDNO:1) with mouse OX40 (SEQ ID NO:3). FIG. 4B depicts the bindingactivity of exemplary anti-OX40 antibody Hu3738 to cell-surfaceexpressed human, murine, or chimeric human-mouse OX40 moleculescontaining mouse cysteine-rich domains (CRDs) swapped out for thecorresponding human regions. Human OX40 is shown as “293s-huOX40,”chimeric human OX40 with murine CRDI is shown as “293s-huOX40-muCRDI,”chimeric human OX40 with murine CRDII is shown as “293s-huOX40-muCRDII,”chimeric human OX40 with murine CRDIII is shown as“293s-huOX40-muCRDIII,” chimeric human OX40 with murine CRDIV is shownas “293s-huOX40-muCRDIV,” chimeric human OX40 with murine CRDII andmurine CRDIII is shown as “293s-huOX40-muCRDII+III,” and murine OX40 isshown as “293s-muOX40.”

FIG. 5 depicts competition for cell surface human OX40 binding byexemplary anti-OX40 antibody Hu3738 or a literature antibody (11D4,18D8, 106-222, 119-122, or 1A7).

FIG. 6A depicts the activation of NF-κB in human OX40-transfected Jurkatreporter cell lines upon treatment with exemplary anti-OX40 antibodyMu3738 or Hu3738, or literature antibody 11D4, 18D8, 106-222, or119-122, or isotype control in the absence of an added cross-linker.FIG. 6B depicts the activation of NF-κB in human OX40-transfected Jurkatreporter cell lines upon treatment with exemplary anti-OX40 antibodyHu3738, literature antibody 1A7, or isotype control in the presence orabsence of an added cross-linker.

FIG. 7 depicts anti-tumor activity of exemplary anti-OX40 antibodyHu3738 in a human PC3 adoptive cell tumor model in NSG mice.

FIG. 8 depicts levels of interleukin-8 (IL-8), granulocyte macrophagecolony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF-α),and interferon-gamma (IFN-γ) in a human peripheral blood mononuclearcell (PBMC) mediated graft-versus-host disease (GVHD) model in NSG mice,after treating the mice with 1 mg/kg Hu3738 or human IgG₁ isotypecontrol once every 7 days for a total of 4 doses.

7. DETAILED DESCRIPTION

The present disclosure concerns antibodies and fragments thereof thatspecifically bind OX40, compositions comprising the antibodies,polynucleotides encoding anti-OX40 antibodies, host cells capable ofproducing the antibodies, methods and compositions useful for making theantibodies, and various methods of using the same.

As will be appreciated by skilled artisans, antibodies and fragmentsthereof are “modular” in nature. Throughout the disclosure, variousspecific embodiments of the various “modules” composing anti-OX40antibodies or binding fragments thereof are described. As specificnon-limiting examples, various specific embodiments of heavy chainvariable domain (V_(H)) complementarity determining regions (CDRs),V_(H) chains, light chain variable domain (V_(L)) CDRs and V_(L) chainsare described. It is intended that all of the specific embodiments maybe combined with each other as though each specific combination wereexplicitly described individually.

7.1. Abbreviations

The antibodies, binding fragments, and polynucleotides described hereinare, in many embodiments, described by way of their respectivepolypeptide or polynucleotide sequences. Unless indicated otherwise,polypeptide sequences are provided in N→C orientation; polynucleotidesequences in 5′→3′ orientation. For polypeptide sequences, theconventional three or one-letter abbreviations for the geneticallyencoded amino acids may be used, as noted in TABLE 1, below.

TABLE 1 Encoded Amino Acid Abbreviations Three Letter One-Letter AminoAcid Abbreviation Abbreviation Alanine Ala A Arginine Arg R AsparagineAsn N Aspartic acid Asp D Cysteine Cys C Glutamic acid Glu E GlutamineGln Q Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu LLysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P SerineSer S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

Certain sequences are defined by structural formulae specifying aminoacid residues belonging to certain classes (e.g., aliphatic,hydrophobic, etc.). The various classes to which the genetically encodedamino acids belong as used herein are noted in TABLE 2, below. Someamino acids may belong to more than one class. Cysteine, which containsa sulfhydryl group, and proline, which is conformationally constrained,are not assigned classes.

TABLE 2 Encoded Amino Acid Classes Class Amino Acids Aliphatic A, I, L,V Aromatic F, Y, W Non-Polar M, A, I, L, V Polar N, Q, S, T Basic H, K,R Acidic D, E Small A, G

7.2. Definitions

Unless otherwise defined herein, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art.

7.3. Anti-OX40 Antibodies and Binding Fragments

OX40 is a co-stimulatory molecule that has a critical role in theenhancement of nascent immune responses and concomitantly acts tosuppress regulatory T cell activity. OX40, also known as CD134 or tumornecrosis factor receptor superfamily 4 (TNFRSF4), is a Type Itransmembrane cell surface member of the tumor necrosis factor (TNF)receptor superfamily transiently expressed on recently activated T cellsand constitutively expressed on activated T regulatory cells. Theextracellular ligand binding domain of OX40 is composed of threecysteine-rich domains (CRD) and a fourth partial CRD (CRDI, CRDII,CRDIII, and CRDIV, respectively). While primarily expressed by activatedCD4+ T cells, OX40 can be expressed on B cells, CD8+ T cells, andnatural killer (NK) and natural killer T (NKT) cells followingactivation. Neutrophils have also been reported to express OX40 andsignaling through OX40 on human neutrophils inhibits apoptotic celldeath. The ligand for OX40 (OX40L), also known as tumor necrosis factorligand superfamily 4 (TNFSF4), CD252 or glycoprotein 34 (gp34), isupregulated by activated antigen-presenting cells and B cells. Ligandbinding to OX40 on antigen-activated T cells results in downstream NF-κBtranslocation and AKT pathway activation. NF-κB translocation leads toupregulation of pro-survival molecules such as Bcl-2, Bcl-XL and cellsurvival. Activating antibodies directed at OX40 are intended at leastin part to enhance antigen-specific immune responses by prolongingactivation and differentiation of T effector cells.

In addition to the impact on antigen activated T effector cells,targeting OX40 expressed by T regulatory cells may also contribute tothe putative mechanism of action. T regulatory cells express high levelsof OX40 within the tumor microenvironment. OX40 activation has beenshown to impact suppressive capacity of T regulatory cells and to leadto the active depletion of OX40 positive T regulatory cells from thetumor microenvironment.

In one aspect, the disclosure concerns antibodies that specifically bindOX40.

As used herein, the term “antibody” (Ab) refers to an immunoglobulinmolecule that specifically binds to a particular antigen—here, OX40. Insome embodiments, the anti-OX40 antibodies of the disclosure bind tohuman OX40 (SEQ ID NO:1) (NCBI Reference Sequence NP003318) and therebymodulate the immune system. The resulting immune system response iscytotoxic to tumor cells. Anti-OX40 antibodies comprise complementaritydetermining regions (CDRs), also known as hypervariable regions, in boththe light chain and the heavy chain variable domains. The more highlyconserved portions of variable domains are called the framework (FR). Asis known in the art, the amino acid position/boundary delineating ahypervariable region of an antibody can vary, depending on the contextand the various definitions known in the art. Some positions within avariable domain may be viewed as hybrid hypervariable positions in thatthese positions can be deemed to be within a hypervariable region underone set of criteria while being deemed to be outside a hypervariableregion under a different set of criteria. One or more of these positionscan also be found in extended hypervariable regions. The disclosureprovides antibodies comprising modifications in these hybridhypervariable positions. The variable domains of native heavy and lightchains each comprise four FR regions, largely by adopting a β-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of the targetbinding site of antibodies. See Kabat et al., Sequences of Proteins ofImmunological Interest (National Institute of Health, Bethesda, Md.1987). As used herein, numbering of immunoglobulin amino acid residuesis done according to the immunoglobulin amino acid residue numberingsystem of Kabat et al. unless otherwise indicated.

The antibodies of the disclosure may be polyclonal, monoclonal,genetically engineered, and/or otherwise modified in nature, includingbut not limited to chimeric antibodies, humanized antibodies, and humanantibodies. In some embodiments, the constant region is an isotypeselected from: IgA (e.g., IgA₁ or IgA₂), IgD, IgE, IgG (e.g., IgG₁,IgG₂, IgG₃ or IgG₄), and IgM. In specific embodiments, an anti-OX40antibody described herein comprises an IgG₁. In other embodiments, theanti-OX40 antibodies comprise an IgG₂ or IgG₄. As used herein, the“constant region” of an antibody includes the natural constant region,allotypes or natural variants, such as D356E and L358M, or A431G inhuman IgG₁. See, e.g., Jefferis and Lefranc, MAbs, 1(4): 332-338(July-August 2009).

The light constant region of an anti-OX40 antibody may be a kappa (κ)light region or a lambda (2) region. A light region can be any one ofthe known subtypes, e.g., λ₁, λ₂, λ₃, or λ₄. In some embodiments, theanti-OX40 antibody comprises a kappa (κ) light region.

The term “monoclonal antibody” as used herein is not limited toantibodies produced through hybridoma technology. A monoclonal antibodyis derived from a single clone, including any eukaryotic, prokaryotic,or phage clone, by any means available or known in the art. Monoclonalantibodies useful with the present disclosure can be prepared using awide variety of techniques known in the art including the use ofhybridoma, recombinant, and phage display technologies, or a combinationthereof. In many uses of the present disclosure, including in vivo useof the anti-OX40 antibodies in humans, chimeric, humanized, or humanantibodies can be used.

The term “chimeric” antibody as used herein refers to an antibody havingvariable sequences derived from a non-human immunoglobulin, such as arat or a mouse antibody, and human immunoglobulin constant regions,typically chosen from a human immunoglobulin template. Methods forproducing chimeric antibodies are known in the art. See, e.g., Morrison,1985, Science 229(4719):1202-7; Oi et al., 1986, BioTechniques4:214-221; Gillies et al., 1985, J. Immunol. Methods 125:191-202; U.S.Pat. Nos. 5,807,715; 4,816,567; and 4,816,397.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins that contain minimal sequences derived from non-humanimmunoglobulin. In general, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin sequence. The humanizedantibody can also comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin consensussequence. Methods of antibody humanization are known in the art. See,e.g., Riechmann et al., 1988, Nature 332:323-7; U.S. Pat. Nos.5,530,101; 5,585,089; 5,693,761; 5,693,762; and U.S. Pat. No. 6,180,370to Queen et al.; EP239400; PCT publication WO 91/09967; U.S. Pat. No.5,225,539; EP592106; EP519596; Padlan, 1991, Mol. Immunol., 28:489-498;Studnicka et al., 1994, Prot. Eng. 7:805-814; Roguska et al., 1994,Proc. Natl. Acad. Sci. 91:969-973; and U.S. Pat. No. 5,565,332.

“Human antibodies” include antibodies having the amino acid sequence ofa human immunoglobulin and include antibodies isolated from humanimmunoglobulin libraries or from animals transgenic for one or morehuman immunoglobulins and that do not express endogenousimmunoglobulins. Human antibodies can be made by a variety of methodsknown in the art including phage display methods using antibodylibraries derived from human immunoglobulin sequences. See U.S. Pat.Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645; WO98/50433; WO 98/24893; WO 98/16654; WO 96/34096; WO 96/33735; and WO91/10741. Human antibodies can also be produced using transgenic micewhich are incapable of expressing functional endogenous immunoglobulinsbut which can express human immunoglobulin genes. See, e.g., PCTpublications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; U.S.Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016;5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598. In addition,companies such as LakePharma, Inc. (Belmont, Calif.) or Creative BioLabs(Shirley, N.Y.) can be engaged to provide human antibodies directedagainst a selected antigen using technology similar to that describedabove. Fully human antibodies that recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach, a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope (see, Jespers et al., 1988, Biotechnology12:899-903).

Also contemplated are anti-OX40 antibody binding fragments. The bindingfragments of the disclosure include those that are capable ofspecifically binding OX40. Examples of antibody binding fragmentsinclude by way of example and not limitation, Fab, Fab′, F(ab′)₂, Fvfragments, single chain Fv (scFv) fragments and single domain fragments.

A Fab fragment contains the constant domain of the light chain and thefirst constant domain (CH1) of the heavy chain. Fab′ fragments differfrom Fab fragments by the addition of a few residues at the carboxylterminus of the heavy chain CH1 domain including one or more cysteinesfrom the antibody hinge region. Fab′ fragments are produced by cleavageof the disulfide bond at the hinge cysteines of the F(ab′)₂ pepsindigestion product. Additional chemical couplings of antibody fragmentsare known to those of ordinary skill in the art. Fab and F(ab′)₂fragments lack the Fragment crystallizable (Fc) region of an intactantibody, clear more rapidly from the circulation of animals, and mayhave less non-specific tissue binding than an intact antibody (see,e.g., Wahl et al., 1983, J. Nucl. Med. 24:316).

As is commonly understood in the art, an “Fc” region is the Fragmentcrystallizable constant region of an antibody not comprising anantigen-specific binding region. In IgG, IgA and IgD antibody isotypes,the Fc region is composed of two identical protein fragments, derivedfrom the second and third constant domains (CH2 and CH3 domains,respectively) of the two heavy chains of an antibody. IgM and IgE Fcregions contain three heavy chain constant domains (CH2, CH3, and CH4domains) in each polypeptide chain.

An “Fv” fragment is the minimum fragment of an antibody that contains acomplete target recognition and binding site. This region consists of adimer of one heavy and one light chain variable domain in a tight,non-covalent association (V_(H)-V_(L) dimer). It is in thisconfiguration that the three CDRs of each variable domain interact todefine a target binding site on the surface of the V_(H)-V_(L) dimer.Often, the six CDRs confer target binding specificity to the antibody.However, in some instances even a single variable domain (or half of anFv comprising only three CDRs specific for a target) can have theability to recognize and bind target, although at a lower affinity thanthe entire binding site.

“Single-chain Fv” or “scFv” antibody binding fragments comprise theV_(H) and V_(L) domains of an antibody, where these domains are presentin a single polypeptide chain. Generally, the Fv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the scFv to form a structure favorable for target binding.

“Single domain fragments” are composed of a single V_(H) or V_(L)domains which exhibit sufficient affinity to OX40. In a specificembodiment, the single domain fragment is camelized (See, e.g.,Riechmann, 1999, Journal of Immunological Methods 231:25-38).

Anti-OX40 antibodies of the disclosure include derivatized antibodies.For example, derivatized antibodies are typically modified byglycosylation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein. Any of numerouschemical modifications can be carried out by known techniques,including, but not limited to, specific chemical cleavage, acetylation,formylation, metabolic synthesis of tunicamycin, etc. Additionally, thederivative can contain one or more non-natural amino acids, e.g., usingambrx technology (See, e.g., Wolfson, 2006, Chem. Biol. 13(10):1011-2).

The anti-OX40 antibodies may be antibodies whose sequences have beenmodified to alter at least one constant region-mediated biologicaleffector function. For example, in some embodiments, an anti-OX40antibody may be modified to reduce at least one constant region-mediatedbiological effector function relative to the unmodified antibody, e.g.,reduced binding to one or more of the Fc receptors (FcγR) such as FcγR1,FcγRIIA, FcγRIIB, FcγRIIIA and/or FcγRIIIB. FcγR binding can be reducedby mutating the immunoglobulin constant region segment of the antibodyat particular regions necessary for FcγR interactions (See, e.g.,Canfield and Morrison, 1991, J. Exp. Med. 173:1483-1491; and Lund etal., 1991, J. Immunol. 147:2657-2662). Reduction in FcγR binding abilityof the antibody can also reduce other effector functions which rely onFcγR interactions, such as opsonization, phagocytosis andantigen-dependent cellular cytotoxicity (“ADCC”). In an illustrativeexample, a variant CH2 domain having a V263L, V273C, V273E, V273F,V273L, V273M, V273S, or V273Y substitution in the CH2 domain of the Fcregion can exhibit reduced affinity to FcγRIIB as compared to thecorresponding wild type constant region.

The anti-OX40 antibody described herein include antibodies that havebeen modified to acquire or improve at least one constantregion-mediated biological effector function relative to an unmodifiedantibody, e.g., to enhance FcγR interactions (See, e.g., US Patent Appl.No. 2006/0134709). For example, an anti-OX40 antibody of the disclosurecan have a constant region that binds FcγR1, FcγRIIA, FcγRIIB, FcγRIIIAand/or FcγRIIIB with greater affinity than the corresponding wild typeconstant region. In an illustrative example, a variant CH2 domain havinga V263L, V273C, V273E, V273F, V273L, V273M, V273S, or V273Y substitutionin the CH2 domain of the Fc region can exhibit greater affinity toFcγRIIIA as compared to the corresponding wild type constant region.

Thus, anti-OX40 antibodies of the disclosure may have alterations inbiological activity that result in increased or decreased opsonization,phagocytosis, or ADCC. Such alterations are known in the art. Forexample, modifications in antibodies that reduce ADCC activity aredescribed in U.S. Pat. No. 5,834,597. An exemplary ADCC lowering variantcorresponds to “mutant 3” (also known as “M3,” shown in FIG. 4 of U.S.Pat. No. 5,834,597) in which residues 234 and 237 (using EU numbering)are substituted with alanines. A mutant 3 (also known as “M3”) variationmay be used in a number of antibody isotypes, e.g., human IgG₂ M3.

Additional substitutions that can modify FcγR binding and/or ADCCeffector function of an anti-OX40 antibody include the K322Asubstitution or the L234A and L235A double substitution in the Fcregion, for example, a human IgG₁ having the L234A/L235A doublesubstitution. See, e.g., Hezareh, et al. J. Virol., 75 (24): 12161-12168(2001).

In some embodiments, the anti-OX40 antibodies have low levels of, orlack, fucose. Antibodies lacking fucose have been correlated withenhanced ADCC activity, especially at low doses of antibody. See Shieldset al., 2002, J. Biol. Chem. 277:26733-26740; Shinkawa et al., 2003, J.Biol. Chem. 278:3466-73. Methods of preparing fucose-less antibodiesinclude growth in rat myeloma YB2/0 cells (ATCC CRL 1662). YB2/0 cellsexpress low levels of FUT8 mRNA, which encodes α-1,6-fucosyltransferase,an enzyme necessary for fucosylation of polypeptides.

Anti-OX40 antibodies can comprise modified (or variant) CH2 domains orentire Fc domains that include amino acid substitutions that increasebinding to FcγRIIB and/or reduced binding to FcγRIIIA as compared to thebinding of a corresponding wild-type CH2 or Fc region. Variant CH2 orvariant Fc domains have been described in U.S. Patent Appl. No.2014/0377253. A variant CH2 or variant Fc domain typically includes oneor more substitutions at position 263, position 266, position 273, andposition 305, wherein the numbering of the residues in the Fc domain isthat of the EU index as in Kabat. In some embodiments, the anti-OX40antibodies comprise one or more substitutions selected from V263L,V266L, V273C, V273E, V273F, V273L, V273M, V273S, V273Y, V305K, andV305W, relative to the wild-type CH2 domain. In specific embodiments,the one or more substitutions of the CH2 domain are selected from V263L,V273E, V273F, V273M, V273S, and V273Y, relative to the CH2 domain of ahuman IgG₁. For example, the one or more substitutions of an IgG₁ CH2domain can be V273E. In another specific embodiment, the anti-OX40antibody of the disclosure comprises a variant IgG₁ CH2 domaincomprising the amino acid substitution V263L.

Other examples of variant CH2 or variant Fc domains that can affordincreased binding to FcγRIIB and/or reduced binding to FcγRIIIA ascompared to the binding of a corresponding wild-type CH2 or Fc regioninclude those found in Vonderheide, et al. Clin. Cancer Res., 19(5),1035-1043 (2013), such as S267E or S267E/L328F in human IgG₁.

In some embodiments, the anti-OX40 antibodies include modifications thatincrease or decrease their binding affinities to the fetal Fc receptor,FcRn, for example, by mutating the immunoglobulin constant regionsegment at particular regions involved in FcRn interactions (see, e.g.,WO 2005/123780). In particular embodiments, an anti-OX40 antibody of theIgG class is mutated such that at least one of amino acid residues 250,314, and 428 of the heavy chain constant region is substituted alone, orin any combinations thereof, such as at positions 250 and 428, or atpositions 250 and 314, or at positions 314 and 428, or at positions 250,314, and 428, with positions 250 and 428 a specific combination. Forposition 250, the substituting amino acid residue can be any amino acidresidue other than threonine, including, but not limited to, alanine,cysteine, aspartic acid, glutamic acid, phenylalanine, glycine,histidine, isoleucine, lysine, leucine, methionine, asparagine, proline,glutamine, arginine, serine, valine, tryptophan, or tyrosine. Forposition 314, the substituting amino acid residue can be any amino acidresidue other than leucine, including, but not limited to, alanine,cysteine, aspartic acid, glutamic acid, phenylalanine, glycine,histidine, isoleucine, lysine, methionine, asparagine, proline,glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine.For position 428, the substituting amino acid residues can be any aminoacid residue other than methionine, including, but not limited to,alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine,histidine, isoleucine, lysine, leucine, asparagine, proline, glutamine,arginine, serine, threonine, valine, tryptophan, or tyrosine. Anexemplary substitution known to modify Fc effector function is the Fcsubstitution M428L, which can occur in combination with the Fcsubstitution T250Q. Additional specific combinations of suitable aminoacid substitutions are identified in Table 1 of U.S. Pat. No. 7,217,797.Such mutations increase binding to FcRn, which protects the antibodyfrom degradation and increases its half-life.

An anti-OX40 antibody may have one or more amino acids inserted into oneor more of its CDRs, for example as described in Jung and Plükthun,1997, Protein Engineering 10:8, 959-966; Yazaki et al., 2004, ProteinEng. Des Sel. 17(5):481-9. Epub 2004 Aug. 17; and U.S. Pat. Appl. No.2007/0280931.

Anti-OX40 antibodies with high affinity for human OX40 (SEQ ID NO:1) maybe desirable for therapeutic and diagnostic uses. Accordingly, thepresent disclosure contemplates antibodies having a high bindingaffinity to human OX40. In specific embodiments, the anti-OX40antibodies bind human OX40 with an affinity of at least about 100 nM,but may exhibit higher affinity, for example, at least about 90 nM, 80nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 7 nM,6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.1 nM, 0.01 nM, or even higher. Insome embodiments, the antibodies bind human OX40 with an affinity in therange of about 1 pM to about 100 nM, or an affinity ranging between anyof the foregoing values, such as but not limited to from about 0.001 to10 nM, 0.001 to 5 nM, 0.01 to 100 nM, 0.01 to 50 nM, 0.01 to 10 nM, 0.01to 5 nM, or 0.01 to 1 nM.

Affinity of anti-OX40 antibodies for human OX40 can be determined usingtechniques well known in the art or described herein, such as forexample, but not by way of limitation, ELISA, isothermal titrationcalorimetry (ITC), surface plasmon resonance, or fluorescentpolarization assay.

Anti-OX40 antibodies generally comprise a heavy chain comprising avariable region (V_(H)) having three complementarity determining regions(“CDRs”) referred to herein (in N→C order) as V_(H) CDR #1, V_(H) CDR#2, and V_(H) CDR #3, and a light chain comprising a variable region(V_(L)) having three complementarity determining regions referred toherein (in N→C order) as V_(L) CDR #1, V_(L) CDR #2, and V_(L) CDR #3.The amino acid sequences of exemplary CDRs, as well as the amino acidsequence of the V_(H) and V_(L) regions of the heavy and light chains ofexemplary anti-OX40 are provided herein. Specific embodiments ofanti-OX40 antibodies include these exemplary CDRs and/or V_(H) and/orV_(L) sequences, as well as antibodies that compete for binding humanOX40 with such antibodies.

In some embodiments, the amino acid sequences of the CDRs of ananti-OX40 antibody have sequences selected from their respective V_(H)and V_(L) CDR sequences in TABLE 3 below:

TABLE 3 Exemplary CDR Sequences CDR Sequence Identifier V_(H) CDR#1:GFTFSRYGMS (SEQ ID NO: 101) GYSIASGYYWN (SEQ ID NO: 111) GFNIKDTYMH(SEQ ID NO: 121) GFSLTSYGVH (SEQ ID NO: 131) V_(H) CDR#2:TINSNGGRTYYPDSVKG (SEQ ID NO: 102) YISYDGSNNYNPSLG (SEQ ID NO: 112)RIDPANGNTKYDPKFQG (SEQ ID NO: 122) VIWSGGSTDYNAAFIS (SEQ ID NO: 132)V_(H) CDR#3: EGITTAYAMDY (SEQ ID NO: 103) TLPYYFDY (SEQ ID NO: 113)GGPAWFVY (SEQ ID NO: 123) EEFDY (SEQ ID NO: 133) V_(L) CDR#1:KASQSVDYDGDSYMH (SEQ ID NO: 104) RASQDISNYLN (SEQ ID NO: 114)V_(L) CDR#2: AASILES (SEQ ID NO: 105) YTSRLHS (SEQ ID NO: 115) YTSRLRS(SEQ ID NO: 125) V_(L) CDR#3: QQSNEDPRT (SEQ ID NO: 106) QQGNTLPLT(SEQ ID NO: 116) QQGNTLPWT (SEQ ID NO: 126) QQGYTLPPT (SEQ ID NO: 136)

Specific exemplary embodiments of anti-OX40 antibodies with the aboveCDRs are described herein. In some embodiments, an anti-OX40 antibodyhas the CDRs according to SEQ ID NOS: 101, 102, 103, 104, 105, and 106.In some embodiments, an anti-OX40 antibody has the CDRs according to SEQID NOS: 111, 112, 113, 114, 115, and 116. In some embodiments, ananti-OX40 antibody has the CDRs according to SEQ ID NOS: 121, 122, 123,114, 125, and 126. In some embodiments, an anti-OX40 antibody has theCDRs according to SEQ ID NOS: 131, 132, 133, 114, 115, and 136.

The CDRs described herein form binding elements within V_(H) and V_(L)chains of anti-OX40 antibodies of the disclosure. TABLES 4 and 5 belowdescribe V_(H) and V_(L) chains corresponding to exemplary anti-OX40antibodies containing the above-described CDRs. The CDRs are underlinedbelow in TABLES 4 and 5. In some embodiments, an anti-OX40 antibodycomprises a V_(H) chain having an amino acid sequence as described inTABLE 4:

TABLE 4 Exemplary V_(H) Sequences V_(H) Sequence Identifier Mu3738EVQLVESGGGLVQPGGSLKLSCAASGFTFSRYGMSWVRQT (SEQ ID NO: 21) V_(H)PDKRLELVATINSNGGRTYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCAREGITTAYAMDYWGQGTSVTVSS Hu3738EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYGMSWVRQA (SEQ ID NO: 22) V_(H).1bPGKGLELVATINSNGGRTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGITTAYAMDYWGQGTTVTVSS Mu3726NVQLQESGPGLVKPSQSLSLTCSVTGYSIASGYYWNWIRQ (SEQ ID NO: 23) V_(H)FPGNKLEWMGYISYDGSNNYNPSLGNRISITRDTSKNQVFLKLNSVTTEDTATYYCVKTLPYYFDYWGQGTTLTVSS Hu3726EVQLQESGPGLVKPSDTLSLTCAVSGYSIASGYYWNWIRQ (SEQ ID NO: 24) V_(H).1aPPGKGLEWMGYISYDGSNNYNPSLGNRITISRDTSKNQVSLKLSSVTAVDTAVYYCVKTLPYYFDYWGQGTTVTVSS Mu3739EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMHWVKQR (SEQ ID NO: 25) V_(H)PEQGLEWIGRIDPANGNTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTDVYYCARGGPAWFVYWGQGTLVTVSA Hu3739EVQLVQSGAEVKKPGSSVKVSCKASGFNIKDTYMHWVRQA (SEQ ID NO: 26) V_(H).1bPGQGLEWIGRIDPANGNTKYDPKFQGRATITADTSTNTAYMELSSLRSEDTAVYYCARGGPAWFVYWGQGTLVTVSS Mu3741QVQLKQSGPGLVQPSQSLSITCTVSGFSLTSYGVHWVRQS (SEQ ID NO: 27) V_(H)PGKGLEWLGVIWSGGSTDYNAAFISRLSISKDNSKSQVFFKMNSLQADDTAIYCCAREEFDYWGQGTTLTVSS Hu3741EVQLVESGGGLVQPGGSLRLSCAVSGFSLTSYGVHWVRQA (SEQ ID NO: 28) V_(H).2bPGKGLEWLGVIWSGGSTDYNAAFISRLTISKDNSKSTVYLQMNSLRAEDTAVYYCAREEFDYWGQGTTVTVSSand a V_(L) chain having an amino acid sequence as described in TABLE 5:

TABLE 5 Exemplary V_(L) Sequences V_(L) Sequence Identifier Mu3738DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMHWY (SEQ ID NO: 31) V_(L)QQKPGQPPKLLIYAASILESGIPARFSGSGSGTDFTLNIH PVEEEDAATYYCQQSNEDPRTFGGGTKLEIKHu3738 DIVMTQSPDSLAVSLGERATINCKASQSVDYDGDSYMHWY (SEQ ID NO: 32) V_(L).1QQKPGQPPKLLIYAASILESGVPDRFSGSGSGTDFTLTIS SLQAEDVAVYYCQQSNEDPRTFGGGTKVEIKMu3726 DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKP (SEQ ID NO: 33) V_(L)DGTVKLLIFYTSRLHSGVPSRFSGGGSGTDYSLTISNLEQ EDIATYFCQQGNTLPLTFGAGTKLELKHu3726 DIQMTQTPSSLSASVGDRVTITCRASQDISNYLNWYQQKP (SEQ ID NO: 34) V_(L).1bGKAPKLLIFYTSRLHSGVPSRFSGSGSGTDYTLTISSLQP EDFATYYCQQGNTLPLTFGQGTKLEIKMu3739 DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKP (SEQ ID NO: 35) V_(L)DGTVKLLIYYTSRLRSGLPSRFSGSGSGTDYSLTISNLEQ EDIATYFCQQGNTLPWTFGGGTKLEIKHu3739 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKP (SEQ ID NO: 36) V_(L).1bGKAPKLLIYYTSRLRSGLPSRFSGSGSGTDYTLTISSLQP EDFATYYCQQGNTLPWTFGGGTKVEIKMu3741 DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWFQQKP (SEQ ID NO: 37) V_(L)DGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQ EDIATYFCQQGYTLPPTFGGGTKLEIKHu3741 DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWFQQKP (SEQ ID NO: 38) V_(L).1cGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQP EDFATYYCQQGYTLPPTFGGGTKVEIK

In some embodiments, an anti-OX40 antibody comprises a V_(H) chainhaving an amino acid sequence according to SEQ ID NO:21, and a V_(L)chain having an amino acid sequence according to SEQ ID NO:31. In someembodiments, an anti-OX40 antibody comprises a V_(H) chain having anamino acid sequence according to SEQ ID NO:23, and a V_(L) chain havingan amino acid sequence according to SEQ ID NO:33. In some embodiments,an anti-OX40 antibody comprises a V_(H) chain having an amino acidsequence according to SEQ ID NO:25, and a V_(L) chain having an aminoacid sequence according to SEQ ID NO:35. In some embodiments, ananti-OX40 antibody comprises a V_(H) chain having an amino acid sequenceaccording to SEQ ID NO:27, and a V_(L) chain having an amino acidsequence according to SEQ ID NO:37.

In some embodiments, an anti-OX40 antibody is suitable foradministration to humans. In a specific embodiment, the anti-OX40antibody is humanized. In some embodiments, an anti-OX40 antibodycomprises a V_(H) chain having an amino acid sequence according to SEQID NO:22, and a V_(L) chain having an amino acid sequence according toSEQ ID NO:32. In some embodiments, an anti-OX40 antibody comprises aV_(H) chain having an amino acid sequence according to SEQ ID NO:24, anda V_(L) chain having an amino acid sequence according to SEQ ID NO:34.In some embodiments, an anti-OX40 antibody comprises a V_(H) chainhaving an amino acid sequence according to SEQ ID NO:26, and a V_(L)chain having an amino acid sequence according to SEQ ID NO:36. In someembodiments, an anti-OX40 antibody comprises a V_(H) chain having anamino acid sequence according to SEQ ID NO:28, and a V_(L) chain havingan amino acid sequence according to SEQ ID NO:38.

Certain mutations of a V_(H) or V_(L) sequence in an anti-OX40 antibodydescribed herein would be understood by a person of skill to affordanti-OX40 antibodies within the scope of the disclosure. Mutations mayinclude amino acid substitutions, additions, or deletions from a V_(H)or V_(L) sequence as disclosed herein while retaining significantanti-OX40 activity. Accordingly, in some embodiments, an anti-OX40antibody comprises a V_(H) sequence having at least 85%, at least 90%,at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99% sequence identity to any one of the V_(H) sequences shownin TABLE 4. An anti-OX40 antibody can comprise a V_(H) sequence havingup to 8, up to 7, up to 6, up to 5, up to 4, up to 3, or up to 2mutations compared with any one of the V_(H) sequences shown in TABLE 4.In some embodiments, an anti-OX40 antibody can comprise a V_(H) sequencehaving 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer mutationscompared with any one of the V_(H) sequences shown in TABLE 4. In someembodiments, an anti-OX40 antibody comprises a V_(L) sequence having atleast 85%, at least 90%, at least 93%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% sequence identity to any one ofthe V_(L) sequences shown in TABLE 5. An anti-OX40 antibody can comprisea V_(L) sequence having up to 8, up to 7, up to 6, up to 5, up to 4, upto 3, or up to 2 mutations compared with any one of the V_(L) sequencesshown in TABLE 5. In some embodiments, an anti-OX40 antibody cancomprise a V_(L) sequence having 5 or fewer, 4 or fewer, 3 or fewer, or2 or fewer mutations compared with any one of the V_(L) sequences shownin TABLE 5.

Full length heavy and light chain amino acid sequences generallycomprise an above-described V_(H) or V_(L) chain linked to anappropriate immunoglobulin constant region, e.g., human IgG₁ or kappalight constant region. Post-translational modifications to the fulllength sequences of an anti-OX40 antibody may occur, such as cleavage ofone or more (e.g., 1, 2, 3, or more) amino acid residues on theC-terminal end of the antibody heavy chain. Such cleavage products maycomprise some or all of the anti-OX40 antibody as expressed.

Accordingly, in some embodiments, an anti-OX40 antibody comprises aheavy chain amino acid sequence as described in TABLE 6:

TABLE 6 Exemplary Heavy Chain Sequences Sequence IdentifierEVQLVESGGGLVQPGGSLRLSCAASGFTFSRYGMSWVR SEQ IDQAPGKGLELVATINSNGGRTYYPDSVKGRFTISRDNAK NO: 41NSLYLQMNSLRAEDTAVYYCAREGITTAYAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

TABLE 6 Exemplary Heavy Chain Sequences Sequence IdentifierEVQLVESGGGLVQPGGSLRLSCAASGFTFSRYGMSWVR SEQ IDQAPGKGLELVATINSNGGRTYYPDSVKGRFTISRDNAK NO: 42NSLYLQMNSLRAEDTAVYYCAREGITTAYAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGEVQLQESGPGLVKPSDTLSLTCAVSGYSIASGYYWNWI SEQ IDRQPPGKGLEWMGYISYDGSNNYNPSLGNRITISRDTSK NO: 43NQVSLKLSSVTAVDTAVYYCVKTLPYYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGKEVQLQESGPGLVKPSDTLSLTCAVSGYSIASGYYWNWI SEQ IDRQPPGKGLEWMGYISYDGSNNYNPSLGNRITISRDTSK NO: 44NQVSLKLSSVTAVDTAVYYCVKTLPYYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGEVQLVQSGAEVKKPGSSVKVSCKASGFNIKDTYMHWVR SEQ IDQAPGQGLEWIGRIDPANGNTKYDPKFQGRATITADTST NO: 45NTAYMELSSLRSEDTAVYYCARGGPAWFVYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGKEVQLVQSGAEVKKPGSSVKVSCKASGFNIKDTYMHWVR SEQ IDQAPGQGLEWIGRIDPANGNTKYDPKFQGRATITADTST NO: 46NTAYMELSSLRSEDTAVYYCARGGPAWFVYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGEVQLVESGGGLVQPGGSLRLSCAVSGFSLTSYGVHWVR SEQ IDQAPGKGLEWLGVIWSGGSTDYNAAFISRLTISKDNSKS NO: 47TVYLQMNSLRAEDTAVYYCAREEFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGKEVQLVESGGGLVQPGGSLRLSCAVSGFSLTSYGVHWVR SEQ IDQAPGKGLEWLGVIWSGGSTDYNAAFISRLTISKDNSKS NO: 48TVYLQMNSLRAEDTAVYYCAREEFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGand a light chain amino acid sequence as described in TABLE 7:

TABLE 7 Exemplary Light Chain Sequences Sequence IdentifierDIVMTQSPDSLAVSLGERATINCKASQSVDYDGDSYMH SEQ IDWYQQKPGQPPKLLIYAASILESGVPDRFSGSGSGTDFT NO: 51LTISSLQAEDVAVYYCQQSNEDPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGECDIQMTQTPSSLSASVGDRVTITCRASQDISNYLNWYQQ SEQ IDKPGKAPKLLIFYTSRLHSGVPSRFSGSGSGTDYTLTIS NO: 52SLQPEDFATYYCQQGNTLPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGECDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQ SEQ IDKPGKAPKLLIYYTSRLRSGLPSRFSGSGSGTDYTLTIS NO: 53SLQPEDFATYYCQQGNTLPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGECDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWFQQ SEQ IDKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTIS NO: 54SLQPEDFATYYCQQGYTLPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGECwherein the underlined amino acids represent the CDRs and the italicizedamino acids represent the constant regions.

In some embodiments, an anti-OX40 antibody comprises a heavy chain aminoacid sequence according to SEQ ID NO:41 or 42, and a light chain aminoacid sequence according to SEQ ID NO:51. In some embodiments, ananti-OX40 antibody comprises a heavy chain amino acid sequence accordingto SEQ ID NO:43 or 44, and a light chain amino acid sequence accordingto SEQ ID NO:52. In some embodiments, an anti-OX40 antibody comprises aheavy chain amino acid sequence according to SEQ ID NO:45 or 46, and alight chain amino acid sequence according to SEQ ID NO:53. In someembodiments, an anti-OX40 antibody comprises a heavy chain amino acidsequence according to SEQ ID NO:47 or 48, and a light chain amino acidsequence according to SEQ ID NO:54.

In some embodiments, an anti-OX40 antibody comprises a heavy chainsequence having at least 85%, at least 90%, at least 93%, at least 95%,at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity to the heavy chain sequence according to any one of SEQ IDNOS:41-48. An anti-OX40 antibody can comprise a heavy chain sequencehaving up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, or up to 2mutations compared with the heavy chain sequence according to any one ofSEQ ID NOS:41-48. In some embodiments, an anti-OX40 antibody cancomprise a heavy chain sequence having 5 or fewer, 4 or fewer, 3 orfewer, or 2 or fewer mutations compared with the heavy chain sequenceaccording to any one of SEQ ID NOS:41-48.

In some embodiments, an anti-OX40 antibody comprises a light chainsequence having at least 85%, at least 90%, at least 93%, at least 95%,at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity to the light chain sequence according to any one of SEQ IDNOS:51-54. An anti-OX40 antibody can comprise a light chain sequencehaving up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, or up to 2mutations compared with the light chain sequence according to any one ofSEQ ID NOS:51-54. In some embodiments, an anti-OX40 antibody cancomprise a light chain sequence having 5 or fewer, 4 or fewer, 3 orfewer, or 2 or fewer mutations compared with the light chain sequenceaccording to any one of SEQ ID NOS:51-54.

Additional post-translational modifications of an anti-OX40 antibody mayinclude glycosylation. Common biantennary complexes can be composed of acore structure having two N-acetylglucosamine (GlcNAc), three mannose,and two GlcNAc residues that are β-1,2 linked to α-6 mannose and α-3mannose to form two antennae. One or more fucose (Fuc), galactose (Gal),high mannose glycans Man-5 or Man-9, bisecting GlcNAc, and sialic acidincluding N-acetylneuraminic acid (NANA) or N-glycolylneuraminic acid(NGNA) residues may be attached to the core. N-linked glycoforms mayinclude G0 (protein having a core biantennary glycosylation structure),G0F (fucosylated G0), G0F GlcNAc, G1 (protein having a coreglycosylation structure with one galactose residue), G1F (fucosylatedG1), G2 (protein having a core glycosylation structure with twogalactose residues), and/or G2F (fucosylated G2).

In some embodiments, the anti-OX40 antibodies compete for binding humanOX40 (SEQ ID NO:1) in in vitro assays with a reference antibody. In someembodiments, the anti-OX40 antibodies compete for binding human OX40 oncells expressing human OX40. The reference antibody may be any of theanti-OX40 antibodies described herein. In some embodiments, thereference antibody is an antibody having a V_(H) according to onedescribed in TABLE 4 and a V_(L) according to one described in TABLE 5.In specific embodiments, the reference antibody is mouse antibodycomprising Mu3726 V_(H) and Mu3726 V_(L) (“Mu3726”), mouse antibodycomprising Mu3738 V_(H) and Mu3738 V_(L) (“Mu3738”), mouse antibodycomprising Mu3739 V_(H) and Mu3739 V_(L) (“Mu3739”), or mouse antibodycomprising Mu3741 V_(H) and Mu3741 V_(L) (“Mu3741”). In someembodiments, the reference antibody is a humanized version of Mu3726,Mu3738, Mu3739, or Mu3741. In certain embodiments, the referenceantibody is a humanized antibody comprising a heavy chain according toSEQ ID NO:41 or 42 and a light chain according to SEQ ID NO:51(“Hu3738”), a humanized antibody comprising a heavy chain according toSEQ ID NO:43 or 44 and a light chain according to SEQ ID NO:52(“Hu3726”), a humanized antibody comprising a heavy chain according toSEQ ID NO:45 or 46 and a light chain according to SEQ ID NO:53(“Hu3739”), or a humanized antibody comprising a heavy chain accordingto SEQ ID NO:47 or 48 and a light chain according to SEQ ID NO:54(“Hu3741”).

The anti-OX40 antibodies described herein generally bind specifically tohuman OX40. Cross reactivity of the antibodies for binding to OX40 fromother species, for example, from monkey, e.g., cynomolgus monkey, mayoffer advantages, such as the ability to test in monkey animal modelsfor biological activity. Such animal model testing may be used to screenanti-OX40 antibodies to select properties related to efficacy, e.g.,favorable pharmacokinetics, or those related to safety, e.g., decreasedhepatic toxicity. In some embodiments, an anti-OX40 antibody binds tocynomolgus OX40 (SEQ ID NO:2) (NCBI Reference Sequence XP005545179) aswell as human OX40. In some embodiments, an anti-OX40 antibody does notbind to mouse OX40 (SEQ ID NO:3) (NCBI Reference Sequence NP037181).

Assays for competition include, but are not limited to, a radioactivematerial labeled immunoassay (RIA), an enzyme-linked immunosorbent assay(ELISA), a sandwich ELISA, fluorescence activated cell sorting (FACS)assays, and surface plasmon resonance assays.

Surface plasmon resonance (SPR) assays allow for direct measurement ofbinding kinetics between two proteins, e.g., a receptor and an antibody,such as human OX40 receptor and an anti-OX40 antibody, without the needfor a reporter signal or tag. Both the equilibrium dissociation constantK_(D), a measure of binding affinity, as well as its two components—thebinding kinetic rate constants, k_(a) (M⁻¹-sec⁻¹) (association constant,k_(on), or “on rate”) and k_(d) (sec⁻¹) (dissociation constant, k_(off),or “off rate”)—can be determined using SPR. The constants are related bythe following equation:

K _(D) =k _(d) /k _(a).

In some embodiments, the anti-OX40 antibodies have a K_(D) of at leastabout 100 nM, but may exhibit higher affinity, for example, at leastabout 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 15nM, 10 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.1 nM, 0.01 nM, oreven higher. In some embodiments, the anti-OX40 antibody has a K_(D) inthe range of about 1 pM to about 100 nM, or an affinity ranging betweenany of the foregoing values, such as but not limited to from about 0.001to 10 nM, 0.001 to 5 nM, 0.01 to 100 nM, 0.01 to 50 nM, 0.01 to 10 nM,0.01 to 5 nM, or about 0.01 to 1 nM.

In some embodiments, an anti-OX40 antibody has a dissociation constantk_(d) no more than about 10 sec⁻¹, for example, no more than about 1,0.5, 0.2, 0.1, 0.05, 0.01, 0.005, 0.001 sec⁻¹, or even lower. In someembodiments, the anti-OX40 antibody has a k_(d) in the range of about0.001 sec⁻¹ to about 10 sec⁻¹, or a k_(d) ranging between any of theforegoing values, such as but not limited to from about 0.01 to 10sec⁻¹, 0.001 to 0.5 sec⁻¹, 0.001 to 0.2 sec⁻¹, 0.001 to 0.1 sec⁻¹, 0.01to 1 sec⁻¹, 0.001 to 0.05 sec⁻¹, or about 0.001 to 1 sec⁻¹.

In some embodiments, an anti-OX40 antibody has an association constantk_(a) at least about 10⁴ M⁻¹-sec⁻¹, for example, at least about 1×10⁴,5×10⁴, 1×10⁵, 5×10⁵, 1×10⁶, 5×10⁶, 1×10⁷ M⁻¹-sec⁻¹, or even greater. Insome embodiments, the anti-OX40 antibody has a k_(d) in the range ofabout 10⁴ M⁻¹-sec⁻¹ to about 10⁷ M⁻¹-sec⁻¹, or a k_(a) ranging betweenany of the foregoing values, such as but not limited to from about 5×10⁴to 1×10⁷ M⁻¹-sec⁻¹, 5×10⁴ to 5×10⁶ M⁻¹-sec⁻¹, or about 1×10⁴ to 5×10⁶M⁻¹-sec⁻¹.

An anti-OX40 antibody of the disclosure may exhibit a K_(D), k_(d), ork_(a) in a range around a binding kinetics constant measured for any oneof the exemplary anti-OX40 antibodies described herein. For example, insome embodiments, an anti-OX40 antibody has a dissociation constantk_(a) in a range of from about 0.01 to about 100-fold, e.g., about 0.1to about 10-fold, or about 0.5 to about 5-fold, the k_(d) of any one ofHu3738, Hu3726, Hu3739, and Hu3741. In some embodiments, an anti-OX40antibody has an association constant k_(a) in a range of from about 0.01to about 100-fold, e.g., about 0.1 to about 10-fold, or about 0.5 toabout 5-fold, the k_(a) of any one of Hu3738, Hu3726, Hu3739, andHu3741.

In conducting an antibody competition assay between a reference antibodyand a test antibody (irrespective of species or isotype), one may firstlabel the reference with a detectable label, such as a fluorophore,biotin or an enzymatic (or even radioactive) label to enable subsequentidentification. In this case, cells expressing human OX40 are incubatedwith unlabeled test antibody, labeled reference antibody is added, andthe intensity of the bound label is measured. If the test antibodycompetes with the labeled reference antibody by binding to anoverlapping epitope, the intensity will be decreased relative to acontrol reaction carried out without test antibody.

In a specific embodiment of this assay, the concentration of labeledreference antibody that yields 80% of maximal binding (“conc_(80%)”)under the assay conditions (e.g., a specified density of cells) is firstdetermined, and a competition assay carried out with 10× conc_(80%) ofunlabeled test antibody and conc_(80%) of labeled reference antibody.

The inhibition can be expressed as an inhibition constant, or K which iscalculated according to the following formula:

K _(i)=IC₅₀/(1+[reference Ab concentration]/K _(d)),

where IC₅₀ is the concentration of test antibody that yields a 50%reduction in binding of the reference antibody and K_(d) is thedissociation constant of the reference antibody, a measure of itsaffinity for human OX40. Antibodies that compete with anti-OX40antibodies disclosed herein can have a K_(i) from 10 pM to 100 nM underassay conditions described herein.

In various embodiments, a test antibody is considered to compete with areference antibody if it decreases binding of the reference antibody byat least about 20% or more, for example, by at least about 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95% or even more, or by a percentageranging between any of the foregoing values, at a reference antibodyconcentration that is 80% of maximal binding under the specific assayconditions used, and a test antibody concentration that is 10-foldhigher than the reference antibody concentration.

A specific assay and assay conditions useful for assessing whether anantibody competes for binding human OX40 with a reference antibody asdescribed herein is provided in Section 8.1.4.

In some embodiments, the anti-OX40 antibodies of the disclosure activatehuman OX40 (SEQ ID NO:1). OX40 receptor activation can occur by a numberof mechanisms, for example, by affording ligand-like activity againstOX40 receptor. In such cases, an anti-OX40 antibody competes for bindingto OX40 receptor with human OX40 ligand (OX40L, CD252;UniProtKB/Swiss-Prot Code P23510.1) (SEQ ID NO:4).

An anti-OX40 antibody of the disclosure can generally activate OX40receptor in the presence of cross-linking. A specific assay and assayconditions useful for assessing whether an anti-OX40 antibody canactivate OX40 receptor, e.g., human OX40 receptor (SEQ ID NO:1), in thepresence of cross-linking is provided in Section 8.1.8. In someembodiments, an anti-OX40 antibody activates human OX40 receptor in thepresence of cross-linking with an EC₅₀ of from about 1 pM to about 500nM, such as but not limited to from about 0.01 to about 300 nM, fromabout 0.01 to about 100 nM, from about 0.01 to about 10 nM, from about0.01 to about 1 nM, from about 0.1 to about 300 nM, from about 0.1 nM toabout 100 nM, from about 1 nM to about 100 nM, or from about 0.1 nM toabout 100 nM. In some embodiments, an anti-OX40 antibody at 100 μg/mLcan activate human OX40 receptor in the presence of cross-linking to anactivity at least about 3-fold, such as from about 3 to about 1000,e.g., about 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, 200, 400, 500, 700,800 or about 1000-fold higher compared with the activity of human OX40receptor in the absence of the anti-OX40 antibody.

Cross-linking can be provided by a number of methods, including additionof exogenous cross-linker, e.g., by antibodies or antibody F(ab′)₂fragments specific for heavy, light or variable regions of human orhumanized antibodies; by soluble or immobilized protein A; by Fcreceptor transfected cell lines; by endogenous Fc receptor expressingcell lines; by directly coating the subject antibodies to plasticsurfaces; by plastic surfaces coated with exogenous cross-linkingantibodies or Fc receptors; or by beads conjugated to any of the above.In an illustrative example, subject antibodies can be conjugated to aprotein such as biotin, and soluble or immobilized avidin orstreptavidin is used as a cross-linker. In another example, in humanlymph nodes in vivo, the activation of OX40 after binding to ananti-OX40 antibody is expected to occur after receptor cross-linkingprovided by endogenous FcγR+antigen-presenting cells.

In some embodiments, an anti-OX40 antibody binds to and activates humanOX40 receptor in the absence of cross-linking. In some embodiments, ananti-OX40 antibody activates OX40 receptor, e.g., human OX40 receptor(SEQ ID NO:1), in the absence of OX40L, e.g., human OX40L (SEQ ID NO:4).A specific assay and assay conditions useful for assessing whether ananti-OX40 antibody can activate OX40 receptor without cross-linking isprovided in Section 8.1.8. In some embodiments, an anti-OX40 antibodyactivates human OX40 receptor without cross-linking with an EC50 of fromabout 1 pM to about 500 nM, such as but not limited to from about 0.01to about 300 nM, from about 0.01 to about 100 nM, from about 0.1 toabout 300 nM, from about 0.1 nM to about 100 nM, from about 1 nM toabout 100 nM, from about 0.1 nM to about 100 nM, from about 1 to about300 nM, from about 1 to about 100 nM, from about 1 to about 50 nM, orfrom about 10 to about 100 nM. In some embodiments, an anti-OX40antibody at 100 μg/mL can activate human OX40 receptor withoutcross-linking to an activity at least about 5-fold, such as from about 5to about 1000, e.g., about 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, 200,300, 400, 500, 700, 800 or about 1000-fold higher compared with theactivity of human OX40 receptor dosed with an equivalent amount ofisotype antibody. In some embodiments, an anti-OX40 antibody at 10 μg/mLcan activate human OX40 receptor without cross-linking to an activity atleast about 3-fold, such as from about 3 to about 300, e.g., about 3, 5,6, 8, 10, 12, 15, 20, 25, 30, 40, 50, 60, 80, 100, 200, or about300-fold higher compared with the activity of human OX40 receptor dosedwith an equivalent amount of isotype antibody. In some embodiments, ananti-OX40 antibody at 1 μg/mL can activate human OX40 receptor withoutcross-linking to an activity at least about 3-fold, such as from about 3to about 150, e.g., about 3, 4, 5, 6, 7, 8, 10, 12, 15, 20, 25, 30, 40,50, 60, 80, 100, or about 150-fold higher compared with the activity ofhuman OX40 receptor dosed with an equivalent amount of isotype antibody.

In some embodiments, an anti-OX40 antibody activates OX40 receptor,e.g., human OX40 receptor (SEQ ID NO:1), at a higher level in thepresence of cross-linking compared to without cross-linking. A specificassay and assay conditions useful for determining the level at which ananti-OX40 antibody can activate OX40 receptor without cross-linking isprovided in Section 8.1.8. The level of activity can be measured, forexample, in terms of EC₅₀ and/or an observed maximal activation. In someembodiments, the anti-OX40 antibody at 100 μg/mL activates OX40receptor, e.g., human OX40 receptor (SEQ ID NO:1), without cross-linkingat from about 20% to about 95% of NF-κB activity, such as about 25%,30%, 40%, 50%, 60%, 70%, 80%, or about 90%, as compared to the NF-κBactivity with cross-linking in an assay according to Section 8.1.8.

In some embodiments, an anti-OX40 antibody activates human OX40 receptorwithout cross-linking with an EC₅₀ of from about 0.1 nM to about 500 nM,such as but not limited to from about 1 nM to about 100 nM, from about0.1 nM to about 100 nM, from about 1 to about 300 nM, from about 1 toabout 100 nM, from about 1 to about 50 nM, or from about 10 to about 100nM, in an assay according to Section 8.1.8. In some such embodiments, ananti-OX40 antibody at 10 μg/mL can activate human OX40 receptor withoutcross-linking to an activity at least about 3-fold, such as from about 3to about 300, e.g., about 3, 5, 6, 8, 10, 12, 15, 20, 25, 30, 40, 50,60, 80, 100, 200, or about 300-fold higher compared with the activity ofhuman OX40 receptor dosed with an equivalent amount of isotype antibody.In some such embodiments, an anti-OX40 antibody activates human OX40receptor in the presence of cross-linking with an EC₅₀ of from about 1pM to about 300 nM, such as but not limited to from about 0.01 to about300 nM, from about 0.01 to about 100 nM, from about 0.01 to about 10 nM,from about 0.01 to about 1 nM, from about 0.1 to about 300 nM, fromabout 0.1 nM to about 100 nM, from about 1 nM to about 100 nM, or fromabout 0.1 nM to about 100 nM, in an assay according to Section 8.1.8. Insome such embodiments, an anti-OX40 antibody can activate human OX40receptor in the presence of cross-linking at a lower EC₅₀, such as fromabout 1.5 to about 100-fold, such as from about 1.5 to about 10-fold,e.g., about 2, 3, 4, 5, 6, 7, 8, 9, or about 10-fold lower, comparedwith the EC₅₀ of antibody 1A7 described in US publication no.2015/0307617 in an assay according to Section 8.1.8.

An anti-OX40 antibody of the invention can activate human OX40 receptorwithout cross-linking with an EC₅₀ of from about 1 nM to about 100 nM inan assay according to Section 8.1.8, and can activate human OX40receptor in the presence of cross-linking at a lower EC₅₀, such as fromabout 1.5 to about 10-fold lower, compared with the EC₅₀ of antibody 1A7described in US publication no. 2015/0307617 in an assay according toSection 8.1.8. Exemplary anti-OX40 antibodies having the above-recitedproperties include Mu3738 and Hu3738 as described in Examples 2 through8 herein.

Generally, OX40 activation upon treatment with an anti-OX40 antibodyresults in a signal transduction, such as an increase in cytokineproduction (e.g., interferon-gamma (IFN-γ)) and/or an increase in cellproliferation, e.g., CD4+ T cell proliferation. In some embodiments, theincrease in IFN-γ production after treatment with 1 μg/mL of ananti-OX40 antibody is from about 1.5 to about 50 times, such as about1.5, 2, 3, 4, 5, 6, 8, 10, 15, 20, 25, 30, 40, or about 50 times thelevel of IFN-γ production after treatment with an equivalent amount ofan isotype antibody. In some embodiments, the increase in CD4+ T cellproliferation after treatment with 1 μg/mL of an anti-OX40 antibody isfrom about 1.5 to about 20 times, such as about 1.5, 2, 3, 4, 5, 6, 8,10, 15, or about 20 times the level of CD4+ T cell proliferation aftertreatment with an equivalent amount of an isotype antibody. Assays fordetermining cytokine levels or for determining cell proliferation levelsare known in the art. A specific assay and assay conditions fordetermining IFN-γ production and/or CD4+ T cell proliferation isprovided herein in Section 8.1.12.

7.4. Polynucleotides Encoding the Anti-OX40 Antibodies, ExpressionSystems and Methods of Making the Same

The present disclosure encompasses nucleic acid molecules encodingimmunoglobulin light and heavy chain genes for anti-OX40 antibodies,vectors comprising such nucleic acids, and host cells capable ofproducing the anti-OX40 antibodies of the disclosure.

An anti-OX40 antibody of the disclosure can be prepared by recombinantexpression of immunoglobulin light and heavy chain genes in a host cell.To express an antibody recombinantly, a host cell is transfected withone or more recombinant expression vectors carrying DNA fragmentsencoding the immunoglobulin light and heavy chains of the antibody suchthat the light and heavy chains are expressed in the host cell and,optionally, secreted into the medium in which the host cells arecultured, from which medium the antibodies can be recovered. Standardrecombinant DNA methodologies are used to obtain antibody heavy andlight chain genes, incorporate these genes into recombinant expressionvectors and introduce the vectors into host cells, such as thosedescribed in Molecular Cloning; A Laboratory Manual, Second Edition(Sambrook, Fritsch and Maniatis (eds), Cold Spring Harbor, N. Y., 1989),Current Protocols in Molecular Biology (Ausubel, F. M. et al., eds.,Greene Publishing Associates, 1989) and in U.S. Pat. No. 4,816,397.

To generate nucleic acids encoding such anti-OX40 antibodies, DNAfragments encoding the light and heavy chain variable regions are firstobtained. These DNAs can be obtained by amplification and modificationof germline DNA or cDNA encoding light and heavy chain variablesequences, for example using the polymerase chain reaction (PCR).Germline DNA sequences for human heavy and light chain variable regiongenes are known in the art (See, e.g., the “VBASE” human germlinesequence database; see also Kabat, E. A. et al., 1991, Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242; Tomlinson etal., 1992, J. Mol. Biol. 22T:116-198; and Cox et al., 1994, Eur. J.Immunol. 24:827-836).

Once DNA fragments encoding anti-OX40 antibody-related V_(H) and V_(L)segments are obtained, these DNA fragments can be further manipulated bystandard recombinant DNA techniques, for example to convert the variableregion genes to full-length antibody chain genes, to Fab fragment genesor to a scFv gene. In these manipulations, a V_(L)- or V_(H)-encodingDNA fragment is operatively linked to another DNA fragment encodinganother protein, such as an antibody constant region or a flexiblelinker. The term “operatively linked,” as used in this context, isintended to mean that the two DNA fragments are joined such that theamino acid sequences encoded by the two DNA fragments remain in-frame.

The isolated DNA encoding the V_(H) region can be converted to afull-length heavy chain gene by operatively linking the V_(H)-encodingDNA to another DNA molecule encoding heavy chain constant regions (CHLCH2, CH3 and, optionally, CH4). The sequences of human heavy chainconstant region genes are known in the art (See, e.g., Kabat, E. A., etal., 1991, Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. The heavy chain constant regioncan be an IgG₁, IgG₂, IgG₃, IgG₄, IgA, IgE, IgM or IgD constant region,but in certain embodiments is an IgG₁ or IgG₄. For a Fab fragment heavychain gene, the V_(H)-encoding DNA can be operatively linked to anotherDNA molecule encoding only the heavy chain CH1 constant region.

The isolated DNA encoding the V_(L) region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the V_(L)-encoding DNA to another DNA moleculeencoding the light chain constant region, CL. The sequences of humanlight chain constant region genes are known in the art (See, e.g.,Kabat, et al., 1991, Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, NIHPublication No. 91-3242) and DNA fragments encompassing these regionscan be obtained by standard PCR amplification. The light chain constantregion can be a kappa or lambda constant region, but in certainembodiments is a kappa constant region. To create a scFv gene, theV_(H)- and V_(L)-encoding DNA fragments are operatively linked toanother fragment encoding a flexible linker, e.g., encoding the aminoacid sequence (Gly₄˜Ser)₃ (SEQ ID NO:60), such that the V_(H) and V_(L)sequences can be expressed as a contiguous single-chain protein, withthe V_(L) and V_(H) regions joined by the flexible linker (See, e.g.,Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl.Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990, Nature348:552-554).

To express the anti-OX40 antibodies of the disclosure, DNAs encodingpartial or full-length light and heavy chains, obtained as describedabove, are inserted into expression vectors such that the genes areoperatively linked to transcriptional and translational controlsequences. In this context, the term “operatively linked” is intended tomean that an antibody gene is ligated into a vector such thattranscriptional and translational control sequences within the vectorserve their intended function of regulating the transcription andtranslation of the antibody gene. The expression vector and expressioncontrol sequences are chosen to be compatible with the expression hostcell used. The antibody light chain gene and the antibody heavy chaingene can be inserted into separate vectors or, more typically, bothgenes are inserted into the same expression vector.

The antibody genes are inserted into the expression vector by standardmethods (e.g., ligation of complementary restriction sites on theantibody gene fragment and vector, or blunt end ligation if norestriction sites are present). Prior to insertion of the anti-OX40antibody-related light or heavy chain sequences, the expression vectorcan already carry antibody constant region sequences. For example, oneapproach to converting the anti-OX40 monoclonal antibody-related V_(H)and V_(L) sequences to full-length antibody genes is to insert them intoexpression vectors already encoding heavy chain constant and light chainconstant regions, respectively, such that the V_(H) segment isoperatively linked to the CH segment(s) within the vector and the V_(L)segment is operatively linked to the CL segment within the vector.Additionally or alternatively, the recombinant expression vector canencode a signal peptide that facilitates secretion of the antibody chainfrom a host cell. The antibody chain gene can be cloned into the vectorsuch that the signal peptide is linked in-frame to the amino terminus ofthe antibody chain gene. The signal peptide can be an immunoglobulinsignal peptide or a heterologous signal peptide (i.e., a signal peptidefrom a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of the disclosure carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif., 1990. It will be appreciated by those skilled in the artthat the design of the expression vector, including the selection ofregulatory sequences may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Suitable regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., theadenovirus major late promoter (AdMLP)) and polyoma. For furtherdescription of viral regulatory elements, and sequences thereof, see,e.g., U.S. Pat. No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 byBell et al., and U.S. Pat. No. 4,968,615 by Schaffner et al.

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the disclosure can carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (See, e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Suitable selectable marker genes include thedihydrofolate reductase (DHFR) gene (for use in DHFR⁻ host cells withmethotrexate selection/amplification) and the neo gene (for G418selection). For expression of the light and heavy chains, the expressionvector(s) encoding the heavy and light chains is transfected into a hostcell by standard techniques. The various forms of the term“transfection” are intended to encompass a wide variety of techniquescommonly used for the introduction of exogenous DNA into a prokaryoticor eukaryotic host cell, e.g., electroporation, lipofection,calcium-phosphate precipitation, DEAE-dextran transfection and the like.

It is possible to express the anti-OX40 antibodies of the disclosure ineither prokaryotic or eukaryotic host cells. In certain embodiments,expression of antibodies is performed in eukaryotic cells, e.g.,mammalian host cells, of optimal secretion of a properly folded andimmunologically active antibody. Exemplary mammalian host cells forexpressing the recombinant antibodies of the disclosure include ChineseHamster Ovary (CHO cells) (including DHFR⁻ CHO cells, described inUrlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220, usedwith a DHFR selectable marker, e.g., as described in Kaufman and Sharp,1982, Mol. Biol. 159:601-621), NSO myeloma cells, COS cells and SP2cells. When recombinant expression vectors encoding antibody genes areintroduced into mammalian host cells, the antibodies are produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibody in the host cells or secretion of theantibody into the culture medium in which the host cells are grown.Antibodies can be recovered from the culture medium using standardprotein purification methods. Host cells can also be used to produceanti-OX40 binding fragments of antibodies, such as Fab fragments or scFvmolecules. It is understood that variations on the above procedure arewithin the scope of the present disclosure. For example, it can bedesirable to transfect a host cell with DNA encoding either the lightchain or the heavy chain (but not both) of an anti-OX40 antibody of thisdisclosure.

Recombinant DNA technology can also be used to remove some or all of theDNA encoding either or both of the light and heavy chains that is notnecessary for binding to human OX40. The molecules expressed from suchtruncated DNA molecules are also encompassed by the antibodies of thedisclosure.

For recombinant expression of an anti-OX40 antibody of the disclosure,the host cell can be co-transfected with two expression vectors of thedisclosure, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors can contain identical selectable markers, or they can eachcontain a separate selectable marker. Alternatively, a single vector canbe used which encodes both heavy and light chain polypeptides.

Once a nucleic acid encoding one or more portions of an anti-OX40antibody has been obtained, further alterations or mutations can beintroduced into the coding sequence, for example to generate nucleicacids encoding antibodies with different CDR sequences, antibodies withreduced affinity to the Fc receptor, or antibodies of differentsubclasses.

The anti-OX40 antibodies of the disclosure can also be produced bychemical synthesis (e.g., by the methods described in Solid PhasePeptide Synthesis, 2^(nd) ed., 1984 The Pierce Chemical Co., Rockford,Ill.). Variant antibodies can also be generated using a cell-freeplatform (See, e.g., Chu et al., Biochemia No. 2, 2001 (Roche MolecularBiologicals) and Murray et al., 2013, Current Opinion in ChemicalBiology, 17:420-426).

Once an anti-OX40 antibody of the disclosure has been produced byrecombinant expression, it can be purified by any method known in theart for purification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. Further, theanti-OX40 antibodies of the present disclosure can be fused toheterologous polypeptide sequences described herein or otherwise knownin the art to facilitate purification.

Once isolated, the anti-OX40 antibody can, if desired, be furtherpurified, e.g., by high performance liquid chromatography (see, e.g.,Fisher, Laboratory Techniques In Biochemistry And Molecular Biology,Work and Burdon, eds., Elsevier, 1980), or by gel filtrationchromatography on a Superdex™ 75 column (Pharmacia Biotech AB, Uppsala,Sweden).

7.5. Pharmaceutical Compositions

The anti-OX40 antibodies described herein may be in the form ofcompositions comprising the antibody and one or more carriers,excipients and/or diluents (all of which are referred to herein as“carriers”), i.e., buffering agents, stabilizing agents, preservatives,isotonifiers, non-ionic detergents, antioxidants, and othermiscellaneous additives. See, Remington's Pharmaceutical Sciences, 16thedition (Osol, ed. 1980). The compositions may be formulated forspecific uses, such as for veterinary uses or pharmaceutical uses inhumans. The form of the composition (e.g., dry powder, liquidformulation, etc.) and the carriers used will depend upon the intendeduses of the antibody and, for therapeutic uses, the mode ofadministration.

For therapeutic uses, the compositions may be supplied as part of asterile, pharmaceutical composition that includes a pharmaceuticallyacceptable carrier. This composition can be in any suitable form(depending upon the desired method of administering it to a patient).The pharmaceutical composition can be administered to a patient by avariety of routes such as intravenously, intratumorally, orintrathecally. The most suitable route for administration in any givencase will depend on the particular antibody, the subject, and the natureand severity of the disease and the physical condition of the subject.Typically, the pharmaceutical composition will be administeredintravenously.

Pharmaceutical compositions can be conveniently presented in unit dosageforms containing a predetermined amount of an anti-OX40 antibodydescribed herein per dose. The quantity of anti-OX40 antibody includedin a unit dose will depend on the disease being treated, as well asother factors as are well known in the art. Such unit dosages may be inthe form of a lyophilized dry powder containing an amount of antibodysuitable for a single administration, or in the form of a liquid. Drypowder unit dosage forms may be packaged in a kit with a syringe, asuitable quantity of carrier and/or other components useful foradministration. Unit dosages in liquid form may be conveniently suppliedin the form of a syringe pre-filled with a quantity of the anti-OX40antibody suitable for a single administration.

The pharmaceutical compositions may also be supplied in bulk formcontaining quantities of anti-OX40 antibody suitable for multipleadministrations.

Pharmaceutical compositions may be prepared for storage as lyophilizedformulations or aqueous solutions by mixing an antibody having thedesired degree of purity with optional pharmaceutically-acceptablecarriers typically employed in the art. Such additives should benontoxic to the recipients at the dosages and concentrations employed.

For example, for intravenous administration, the composition may be inthe form of a lyophilized powder that, upon reconstitution with sterilewater or other solution suitable for injection or infusion (for example,0.9% saline, Ringer's solution, lactated Ringer's solution, etc.)provides an aqueous composition.

7.6. Methods of Use

7.6.1. Therapeutic Benefit

Data provided herein demonstrate that the disclosed anti-OX40 antibodiesactivate OX40 receptor in the presence of cancer cells and exert potentanticancer activity against cancer in vivo. Accordingly, the anti-OX40antibodies and/or pharmaceutical compositions comprising the anti-OX40antibodies may be used therapeutically to treat cancers.

In some embodiments, the cancer is a solid tumor. Solid tumors that maybe treated with the anti-OX40 antibody include bladder cancer, breastcancer (e.g., triple negative breast cancer), head and neck cancer,kidney cancer (e.g., renal cell carcinoma), liver cancer (e.g.,hepatocellular carcinoma, cholangiocarcinoma), lung cancer (e.g.,non-small cell lung cancer, mesothelioma, small cell lung cancer),melanoma (e.g., unresectable or metastatic melanoma, advanced malignantmelanoma), skin cancer (e.g., Merkel cell carcinoma), ovarian cancer,gastric cancer, and tumors with evidence of DNA mismatch repairdeficiency. The cancer may be comprised of tumors containingOX40-expressing cells; comprised of tumors, some of which containOX40-expressing cells and some of which do not; or comprised of tumorslacking OX40-expressing cells. The cancer may be newly diagnosed andnaïve to treatment, or may be relapsed, refractory, or relapsed andrefractory, or a metastatic form of a solid tumor. In some embodiments,the solid tumor is naïve to a PD-1 or PD-L1 targeting agent. In otherembodiments, the solid tumor is relapsed or refractory after treatmentwith a PD-1 or PD-L1 targeting agent. In some embodiments, the solidtumor is selected from bladder cancer, breast cancer, head and neckcancer, kidney cancer, lung cancer, melanoma, and gastric cancer. Insome embodiments, the solid tumor is selected from: melanoma (e.g.,unresectable or metastatic melanoma), lung cancer (e.g., non-small celllung cancer), and renal cell carcinoma (e.g., advanced renal cellcarcinoma). In some embodiments, the solid tumor is selected from triplenegative breast cancer, ovarian cancer, hepatocellular carcinoma,gastric cancer, small cell lung cancer, mesothelioma,cholangiocarcinoma, Merkel cell carcinoma and tumors with evidence ofDNA mismatch repair deficiency. In certain embodiments, the lung canceris metastatic non-small cell lung cancer with progression on or afterplatinum-based chemotherapy. In certain embodiments, the lung cancer islocally advanced or metastatic non-small cell lung cancer that hasfailed platinum-based therapy and therapy with a PD-1 or PD-L1 targetingagent. In certain embodiments, the head and neck cancer is recurrentsquamous cell head and neck carcinoma that is not a candidate forcurative treatment with local or systemic therapy, or metastatic(disseminated) head and neck squamous cell carcinoma of the oral cavity,oropharynx, hypopharynx, and larynx that is considered incurable bylocal therapies.

As discussed above, the presently disclosed anti-OX40 antibodiesmodulate an immunological response. Accordingly, patients havingcompromised immune systems may be excluded from treatment. In someembodiments, a patient is excluded after meeting one or more of thefollowing criteria: (1) Active or prior documented autoimmune disease(including, but not limited to, inflammatory bowel disease, celiacdisease, Wegener syndrome) within the past 2 years. (Subjects withchildhood atopy or asthma, vitiligo, alopecia, Hashimoto syndrome,Grave's disease, or psoriasis not requiring systemic treatment (withinthe past 2 years) are not excluded); (2) History of primaryimmunodeficiency, bone marrow transplantation, chronic lymphocyticleukemia, solid organ transplantation, or previous clinical diagnosis oftuberculosis; (3) History of a coagulopathy or a platelet disorder; (4)Confirmed positive test results for human immunodeficiency virus (HIV),or subjects with chronic or active hepatitis B or C. (Subjects who havea history of hepatitis B or C who have documented cures after anti-viraltherapy may be enrolled); (5) Prior grade >3 immune-mediatedneurotoxicity or pneumonitis while receiving immunotherapy (includingbut not limited to agents directed against CTLA-4, PD-L1, or PD-1). Inaddition, any other prior grade >3 immune-mediated adverse event whilereceiving immunotherapy that has not resolved or become asymptomaticwithin 3 months; (6) Receipt of live, attenuated vaccine within 28 daysprior to the first dose of the anti-OX40 antibody.

An anti-OX40 antibody of the disclosure may be administered alone(monotherapy) or adjunctive to, or with, other anti-cancer therapiesand/or targeted or non-targeted anti-cancer agents. When administered asan anti-OX40 monotherapy, one or more antibodies may be used. Whetheradministered as monotherapy or adjunctive to, or with, other therapiesor agents, an amount of anti-OX40 antibody is administered such that theoverall treatment regimen provides therapeutic benefit.

By therapeutic benefit is meant that the use of anti-OX40 antibodies totreat cancer in a patient results in any demonstrated clinical benefitcompared with no therapy (when appropriate) or to a known standard ofcare. Clinical benefit can be assessed by any method known to one ofordinary skill in the art. In one embodiment, clinical benefit isassessed based on objective response rate (ORR) (determined using RECISTversion 1.1), duration of response (DOR), progression-free survival(PFS), and/or overall survival (OS). In some embodiments, a completeresponse indicates therapeutic benefit. In some embodiments, a partialresponse indicates therapeutic benefit. In some embodiments, stabledisease indicates therapeutic benefit. In some embodiments, an increasein overall survival indicates therapeutic benefit. In some embodiments,therapeutic benefit constitutes an improvement in time to diseaseprogression and/or an improvement in symptoms or quality of life. Inother embodiments, therapeutic benefit does not translate to anincreased period of disease control, but rather a markedly reducedsymptom burden resulting in improved quality of life. As will beapparent to those of skill in the art, a therapeutic benefit may beobserved using the anti-OX40 antibodies alone (monotherapy) oradjunctive to, or with, other anti-cancer therapies and/or targeted ornon-targeted anti-cancer agents.

Typically, therapeutic benefit is assessed using standard clinical testsdesigned to measure the response to a new treatment for cancer. Toassess the therapeutic benefits of the anti-OX40 antibodies describedherein one or a combination of the following tests can be used: (1) theResponse Evaluation Criteria In Solid Tumors (RECIST) version 1.1, (2)immune-related RECIST (irRECIST), (3) the Eastern Cooperative OncologyGroup (ECOG) Performance Status, (4) immune-related response criteria(irRC), (5) disease evaluable by assessment of tumor antigens, (6)validated patient reported outcome scales, and/or (7) Kaplan-Meierestimates for overall survival and progression free survival.

Assessment of the change in tumor burden is an important feature of theclinical evaluation of cancer therapeutics. Both tumor shrinkage(objective response) and time to the development of disease progressionare important endpoints in cancer clinical trials. Standardized responsecriteria, known as RECIST (Response Evaluation Criteria in SolidTumors), were published in 2000. An update (RECIST 1.1) was released in2009. RECIST criteria are typically used in clinical trials whereobjective response is the primary study endpoint, as well as in trialswhere assessment of stable disease, tumor progression or time toprogression analyses are undertaken because these outcome measures arebased on an assessment of anatomical tumor burden and its change overthe course of the trial. TABLE 8 provides the definitions of theresponse criteria used to determine objective tumor response to a studydrug, such as the anti-OX40 antibodies described herein.

TABLE 8 Response Criteria Complete Response Disappearance of all targetlesions. Any pathological lymph nodes (CR) (whether target ornon-target) must have reduction in short axis to <10 mm. PartialResponse At least a 30% decrease in the sum of diameters of targetlesions, taking as (PR) reference the baseline sum diameters.Progressive Disease At least a 20% increase in the sum of diameters oftarget lesions, taking as (PD) reference the smallest sum on study (thisincludes the baseline sum if that is the smallest on study). In additionto the relative increase of 20%, the sum must also demonstrate anabsolute increase of at least 5 mm. (Note: the appearance of one or morenew lesions is also considered progression). Stable Disease Neithersufficient shrinkage to qualify for PR nor sufficient increase to (SD)qualify for PD, taking as reference the smallest sum diameters while onstudy.

Secondary outcome measures that can be used to determine the therapeuticbenefit of the anti-OX40 antibodies described herein include, ObjectiveResponse Rate (ORR), Progression Free Survival (PFS), Overall Survival(OS), Duration of Overall Response (DOR), and Depth of Response (DpR).ORR is defined as the proportion of the participants who achieve acomplete response (CR) or partial response (PR). PFS is defined as thetime from the first dose date of an anti-OX40 antibody to either diseaseprogression or death, whichever occurs first. OS is defined as thelength of time from either the date of diagnosis or the start oftreatment for a disease, that patients diagnosed with the disease arestill alive. DOR is defined as the time from the participant's initialCR or PR to the time of disease progression. DpR is defined as thepercentage of tumor shrinkage observed at the maximal response pointcompared to baseline tumor load. Clinical endpoints for both ORR and PFScan be determined based on RECIST 1.1 criteria described above.

Additional criteria that may be used for clinical evaluation specific tocancer patients undergoing immune therapy treatment include thestandardized immune-related RECIST (irRECIST) criteria. See, e.g.,Nishino, M. et al. Eur. J. Radiol., 84(7), pages 1259-1268 (2015 July).These guidelines modified the RECIST 1.1 criteria above withconsideration of potential immunomodulatory effects. TABLE 9 providesthe definitions of the response criteria used to determine objectivetumor response to an immunomodulatory drug, such as the anti-OX40antibodies described herein.

TABLE 9 Response Criteria Complete Response Complete disappearance ofall measurable and non-measurable lesions. (irCR) Lymph nodes mustdecrease to <10 mm in short axis. Partial Response Decrease of ≥30% intotal measured tumor burden relative to baseline, (irPR) non-targetlesions are irNN, and no unequivocal progression of new non- measurablelesions Progressive Disease At least a 20% increase and at least 5 mmabsolute increase in TMTB (irPD) compared to nadir, or irPD fornon-target or new non-measurable lesions. Confirmation of progression isrecommended at least 4 weeks after the first irPD assessment. Non-irCRor non- No target disease was identified at baseline and at follow-upthe patient irPD (irNN) fails to meet criteria for irCR or irPD StableDisease Neither sufficient shrinkage to qualify for irPR nor sufficientincrease to (irSD) qualify for irPD, taking as reference the smallestsum diameters while on study. irNE Used in exceptional cases whereinsufficient data exists.

The ECOG Scale of Performance Status shown in TABLE 10 is used todescribe a patient's level of functioning in terms of their ability tocare for themselves, daily activity, and physical ability. The scale wasdeveloped by the Eastern Cooperative Oncology Group (ECOG), now part ofthe ECOG-ACRIN Cancer Research Group, and published in 1982.

TABLE 10 Grade ECOG Performance Status 0 Fully active, able to carry onall pre-disease performance without restriction 1 Restricted inphysically strenuous activity but ambulatory and able to carry out workof a light or sedentary nature, e.g., light house work, office work 2Ambulatory and capable of all selfcare but unable to carry out any workactivities; up and about more than 50% of waking hours 3 Capable of onlylimited selfcare; confined to bed or chair more than 50% of waking hours4 Completely disabled; cannot carry on any selfcare; totally confined tobed or chair 5 Dead

Another set of criteria that can be used to characterize fully and todetermine response to immunotherapeutic agents, such as antibody-basedcancer therapies, is the immune-related response criteria (irRC), whichwas developed for measurement of solid tumors in 2009, and updated in2013 (Wolchok, et al. Clin. Cancer Res. 2009; 15(23): 7412-7420 andNishino, et al. Clin. Cancer Res. 2013; 19(14): 3936-3943). The updatedirRC criteria are typically used to assess the effect of animmunotherapeutic agent, such as an anti-OX40 antibody described herein,on tumor burden, and defines response according to TABLE 11.

TABLE 11 Response Criteria Complete Response Disappearance of all targetlesions in two consecutive observations not less (CR) than 4 weeks apartPartial Response At least a 30% decrease in the sum of the longestdiameters of target (PR) lesions, taking as reference the baseline sumdiameters. Progressive Disease At least a 20% increase in the sum ofdiameters of target lesions, taking as (PD) reference the smallest sumon study (this includes the baseline sum if that is the smallest onstudy). (Note: the appearance of one or more new lesions is notconsidered progression. The measurement of new lesions is included inthe sum of the measurements). Stable Disease Neither sufficientshrinkage to qualify for PR nor sufficient increase to (SD) qualify forPD, taking as reference the smallest sum diameters while on study.

One exemplary therapeutic benefit resulting from the use of anti-OX40antibodies described herein to treat solid tumors, whether administeredas monotherapy or adjunctive to, or with, other therapies or agents, isa complete response. Another exemplary therapeutic benefit resultingfrom the use of anti-OX40 antibodies to treat solid tumors, whetheradministered as monotherapy or adjunctive to, or with, other therapiesor agents, is a partial response.

Validated patient reported outcome scales can also be used to denoteresponse provided by each patient through a specific reporting system.Rather than being disease focused, such outcome scales are concernedwith retained function while managing a chronic condition. Onenon-limiting example of a validated patient reported outcome scale isPROMIS® (Patient Reported Outcomes Measurement Information System) fromthe United States National Institutes of Health. For example, PROMIS®Physical Function Instrument for adult cancer patients can evaluateself-reported capabilities for the functioning of upper extremities(e.g., dexterity), lower extremities (e.g., walking or mobility), andcentral regions (e.g., neck, back mobility), and includes routine dailyactivities, such as running errands.

Kaplan-Meier curves (Kaplan and Meier, J. Am. Stat. Assoc. 1958;53(282): 457-481) can also be used to estimate overall survival andprogression free survival for cancer patients undergoing anti-OX40antibody therapy in comparison to standard of care.

7.6.2. Adjunctive Therapies

The anti-OX40 antibodies may be used adjunctive to, or with, otheragents or treatments having anti-cancer properties, including standardof care therapies, such as an anti-PD-1 antibody therapy. When usedadjunctively, the anti-OX40 antibody and other agent(s) may beformulated together in a single, combination pharmaceutical formulation,or may be formulated and administered separately, either on a singlecoordinated dosing regimen or on different dosing regimens. Agentsadministered adjunctive to or with the anti-OX40 antibodies willtypically have complementary activities to the anti-OX40 antibodies suchthat the antibodies and other agents do not adversely affect each other.

7.7. Dosages and Administration Regimens

The amount of anti-OX40 antibodies administered will depend upon avariety of factors, including but not limited to, the particular type ofcancer treated, the stage of the cancer being treated, the mode ofadministration, the frequency of administration, the desired therapeuticbenefit, and other parameters such as the age, weight and othercharacteristics of the patient, etc. Determination of dosages effectiveto provide therapeutic benefit for specific modes and frequency ofadministration is within the capabilities of those skilled in the art.

Dosages effective to provide therapeutic benefit may be estimatedinitially from in vivo animal models. Suitable animal models for a widevariety of diseases are known in the art.

The anti-OX40 antibodies disclosed herein may be administered by anyroute appropriate to the condition to be treated. In some embodiments,the anti-OX40 antibody is any one of the humanized antibodies with aheavy chain having an amino acid sequence according to any one of SEQ IDNOS:41-48, and a light chain having an amino acid sequence according toany one of SEQ ID NO:51-54. In certain embodiments, the anti-OX40antibody has a heavy chain having an amino acid sequence according toSEQ ID NO:41 or 42, and a light chain having an amino acid sequenceaccording to SEQ ID NO:51. An anti-OX40 antibody will typically beadministered parenterally, i.e., infusion, intravenous (IV),intrathecal, bolus, intratumoral injection or epidural (Shire et al.,2004, J. Pharm. Sciences 93(6):1390-1402). In one embodiment, ananti-OX40 antibody is provided as a lyophilized powder in a vial. Priorto administration, the lyophilized powder is reconstituted with sterilewater for injection (SWFI) or other suitable medium to provide asolution containing anti-OX40 antibody. In some embodiments, theresulting reconstituted solution is further diluted with saline or othersuitable medium for infusion and administered via an IV infusion onceevery two weeks, i.e., every 13, 14 or 15 days.

In some embodiments, the anti-OX40 antibody is administered as an IVinfusion once every two weeks at 0.01 mg/kg, 0.1 mg/kg, 1.0 mg/kg, or3.0 mg/kg.

When administered adjunctive to or with other agents, such as otherchemotherapeutic agents, the anti-OX40 antibodies may be administered onthe same schedule as the other agent(s), or on a different schedule.When administered on the same schedule, the anti-OX40 antibody may beadministered before, after, or concurrently with the other agent.

As will be appreciated by those of skill in the art, the recommendeddosages for the various agents described above may need to be adjustedto optimize patient response and maximize therapeutic benefit.

8. EXAMPLES

The following Examples, which highlight certain features and propertiesof the exemplary embodiments of the anti-OX40 antibodies describedherein are provided for purposes of illustration, and not limitation.

Example 1: Materials and Methods

8.1.1. Anti-OX40 Antibody Binding to Human OX40 by ELISA

Immunolon 4×HB 96-well plates (Thermo Scientific) were coated with 1μg/mL of human OX40-FC (R&D Systems) at 4° C. overnight. Plates wereblocked with phosphate-buffered saline (PBS) containing 1% bovine serumalbumin (BSA) for 30 minutes at room temperature and then washed threetimes with PBST (PBS with 0.1% Tween 20) using a plate washer.OX40-coated plates were then incubated with indicated concentrations oftest antibody at room temperature for one hour. Plates were washed fourtimes with PBST and then incubated for 1 hour at room temperature with100 μL of goat anti-human Fab fragment specific-Biotin (JacksonImmunoResearch) prepared to a dilution of 1:5000 in PBS containing 1%BSA. Plates were then washed five times in PBST and 100 μL of a 1:1000dilution of streptavidin-horseradish peroxidase (HRP) (ThermoScientific) was added to each well and incubated for 30 minutes at roomtemperature. Plates were subsequently washed five times in PBST and 100μL of TMB One component (Surmodics) were added to each well andincubated at room temperature (RT) until color developed (approximately5-10 minutes). Optical density (OD) was read at 650 nm (MolecularDevices Spectromax190).

8.1.2. Anti-OX40 Antibody Binding to Cynomolgus Monkey OX40 by ELISA

Immunolon 4×HB 96-well plates (Thermo Scientific) were coated with 1μg/mL of Cyno OX40-Fc fusion at 4° C. overnight. Plates were blockedwith PBS containing 1% bovine serum albumin (BSA) for 30 minutes at RTand then washed three times with PBST (PBS with 0.1% Tween 20).OX40-coated plates were then incubated with indicated concentrations ofanti-OX40 antibody at room temperature for one hour. Plates were washedfour times with PBST and then incubated for 1 hour at room temperaturewith 100 μL of goat anti-human FAB fragment specific-Biotin (JacksonImmunoResearch) prepared to a dilution of 1:5000 in PBS containing 1%BSA. Plates were then washed five times in PBST and 100 μL of a 1:1000dilution of streptavidin-HRP (Thermo Scientific) was added to each welland incubated for 30 minutes at room temperature. Plates were thenwashed five times in PBST and 100 μL of TMB One component (Surmodics)were added to each well and incubated at room temperature until colordeveloped (approximately 5-10 minutes). Optical density (OD) was read at650 nm (Molecular Devices Spectromax190).

8.1.3. Anti-OX40 Antibody Binding to Rhesus OX40 by Flow Cytometry

Rhesus macaque (Macaca mulatta) OX40 is identical to cynomolgus monkey(Macaca fascicularis) OX40 (SEQ ID NO:2) at the amino acid level. A 293NF-κB reporter cell line expressing rhesus OX40 was cultured inDulbecco's modified Eagle media (DMEM) containing 10% fetal bovine serum(FBS) and Penicillin/Streptomycin. For the binding assay, cells wereresuspended at 5 million cells per mL. 50 μL (250,000 cells)/well weretransferred to each well of a 500 μL polypropylene 96-well plate (Nunc).A 2× stock of test anti-OX40 antibody or isotype control monoclonalantibody was prepared in a separate dilution plate at 666, 333, 111,37.03, 12.34, 4.11, 0.457, 0.152, 0.0508, 0.0169, 0.00564 nM in culturemedia. The monoclonal antibodies (50 μL/well) were transferred intorespective wells of the assay plate. Cells were incubated with theprimary antibodies for 30 minutes at 4° C. and washed twice with 250μL/well of PBS by centrifuging at 800 rpm for 3 minutes. Bound antibodywas detected with Cy5-Donkey anti-human IgG (H+L) (JacksonImmunoResearch) diluted to 2 μg/mL (50 μL/well) in PBS for 30 minutes at4° C. Cells were washed once with 250 μL/well of PBS, resuspended in PBScontaining 1% Formaldehyde and analyzed on a dual laser FACSCalibur(Becton Dickinson).

8.1.4. Anti-OX40 Antibody Binding Affinity to Human and Rhesus OX40 bySurface Plasmon Resonance

The binding kinetics of an anti-OX40 antibody to recombinant solubleOX40 ECD (extracellular domain) were determined by surface plasmonresonance-based measurements made on a Biacore T200 instrument (GEHealthcare) at 25° C. using an anti-Fc capture assay approach.Recombinant extracellular domains (ECDs) of human OX40 (residues 1-216)and rhesus macaque OX40 (residues 28-214) were purchased (CreativeBiomart) and further purified by gel filtration using Superdex200 (GEHealthcare) in 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid(HEPES), pH 7.4, 150 mM NaCl, 3 mM ethylenediaminetetraacetic acid(EDTA). Rhesus macaque (Macaca mulatta) OX40 is identical to cynomolgusmonkey (Macaca fascicularis) OX40 (SEQ ID NO:2) at the amino acid level.Chip preparation and binding kinetic measurements were made in the assaybuffer HBS-EP+(10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Tween20). For anti-Fc capture chip preparation, approximately 2000 ResonanceUnits (RU) of goat anti-human IgG Fc polyclonal antibody (Thermo FisherScientific Inc.), diluted to 25 μg/mL in 10 mM sodium acetate (pH 4.5),was directly immobilized across a CMS biosensor chip using a standardamine coupling kit according to manufacturer's instructions andprocedures. Unreacted moieties on the biosensor surface were blockedwith ethanolamine. For binding kinetics measurements each assay cycleconsisted of the following steps: 1) capture of test anti-OX40 antibodyon test surface only; 2) analyte injection (OX40 ECD or buffer only)over both reference and test surface, 240 μL at 80 μL/min, after whichthe dissociation was monitored for 900 seconds at 80 μL/min; 3)regeneration of capture surface by 10 mM Glycine-HCl, pH 1.5 injectionsover both reference and test surface. During the assay, all measurementswere referenced against the capture surface alone (i.e., with nocaptured test antibody) and buffer-only injections were used for doublereferencing. OX40 injections ranged in concentration from 900 nM or 300nM to 11.11 nM in a randomized 9- or 3-fold dilution series,respectively. Data were processed and fitted globally to a 1:1 bindingmodel using Biacore T200 Evaluation software to determine the bindingkinetic rate constants, k_(a) (M⁻¹s⁻¹) and k_(d) (s⁻¹), and theequilibrium dissociation constant K_(D) (M).

8.1.5. OX40 Ligand Blocking with Anti-OX40 Antibody

Jurkat cells stably transfected with human OX40 cultured at 2×10⁵cells/well were simultaneously incubated with 0.2 μg/mL test anti-OX40antibody and a titration of soluble human OX40L (R&D systems) in PBScontaining 1% BSA in a round bottom 96-well plate for 30 minutes at RT.Cells were washed twice and incubated for an additional 30 minutes with100 μL of 1:500 dilution of goat-anti-human Fc PE per well (JacksonImmunoResearch). Cells were then washed twice and acquired usingFACSCanto (BD Biosciences), and analyzed using FACSDiva.

8.1.6. Anti-OX40 Antibody Binding to Cell Surface Expressed Human OX40

A Jurkat NF-κB reporter cell line expressing human OX40 protein wascultured in DMEM containing 10% FBS and penicillin/streptomycin(pen/strep). For the binding assay, each cell line was resuspended at 5million cells per mL. 50 μL (250,000 cells)/well were transferred toeach well of a 500 μL polypropylene 96-well plate (Nunc). A 2× stock oftest anti-OX40 antibody or isotype control mAb was prepared in aseparate dilution plate at 666, 333, 111, 37.03, 12.34, 4.11, 0.457,0.152, 0.0508, 0.0169, 0.00564 nM in culture media. Each antibody (50μL/well) was transferred into respective wells of the assay plate. Cellswere incubated with the test anti-OX40 antibody or isotype controlantibodies for 30 minutes at 4° C. and washed twice with 250 μL/well ofPBS by centrifuging at 800 rpm for 3 minutes. Bound antibody wasdetected with Cy5-Donkey anti-human IgG (H+L) (Jackson ImmunoResearch)diluted to 2 μg/mL (50 μL/well) in PBS for 30 minutes at 4° C. Cellswere washed once with 250 μL/well of PBS, resuspended in PBS containing1% Formaldehyde and analyzed on a dual laser FACSCalibur (BectonDickinson).

8.1.7. Anti-OX40 Antibody Binding to Chimeric OX40 Receptor

293s-based transfectants were generated to express chimeric versions ofthe human OX40 molecule with mouse OX40 cysteine-rich domains (CRDs)individually swapped into corresponding human CRDs. After G418selection, surviving cells were sorted for expression on the MoFlo flowcytometer (Beckman): 293s-huOX40, 293s-huOX40-muCRD1,293s-huOX40-muCRDII, 293s-huOX40-muCRDIII, 293s-huOX40-muCRDIV,293s-huOX40-muCRDII+III and 293s-muOX40. A total of 2×10⁵ of each 293sOX40 chimeric transfectant cells were added per well into 500 μLpolypropylene 96-well plates (Nunc). After plating cells, 50 μL ofHu3738 or isotype control antibody at 2 μg/mL were added tocorresponding wells in duplicate for each cell line and allowed toincubate on ice for 30 minutes. Following incubation, 200 μL ofDulbecco's Phosphate Buffered Saline (DPBS) was added into each well andplates were spun down at 1000 rpm for three minutes. Supernatants fromeach well were removed and 50 μL of Cy5-Donkey anti-Human IgG (JacksonImmunoResearch) secondary antibody was added at a 1:250 dilution, whichwas then incubated for 30 minutes on ice in the dark. Following theincubation period, 200 μL of DPBS was added prior to spinning down theplate at 1000 rpm for three minutes. Supernatants were removed and eachwell was re-suspended with 100 μL of DPBS+1% Formaldehyde. Samples wereanalyzed on the dual laser FACSCalibur flow cytometer (BectonDickinson).

8.1.8. NF-κB Fluorescence Reporter Activity for Human and Rhesus OX40

Jurkat-NF-κB-huOX40 and 293-NF-κB-RhOX40, NF-κB reporter cell linesexpressing the human and rhesus OX40 proteins, respectively, weremaintained in culture media comprising DMEM containing 10% FBS andpenicillin/streptomycin (100 U/mL). For the NF-κB reporter assay, theJurkat-NF-κB-huOX40 cell line was resuspended in growth media (identicalto culture media) at 1 million/mL (final 50,000 cells/well) and293-NF-κB-RhOX40 cell line was resuspended in growth media at 0.5million/mL (final 25,000 cells/well). 50 μL/well were transferred to theinner 60 wells of a white/clear bottom 96-well assay plate (Costar3903). A 3× stock of the following antibodies were made in a separateU-bottom 96-well dilution plate (Becton Dickinson): anti-PD-1 antibodyused as a negative control antibody, and anti-OX40 antibody. Thedilution series to test the activity of the antibodies without exogenouscross-linker included 2000, 500, 125, 31.25, 7.812, 1.953, 0.488, 0.122,0.0305, 0.00762 nM in culture media. The dilution series to test theeffect of cross-linker on the activity of the anti-OX40 antibodyincluded 200, 50, 12.5, 3.125, 0.7812, 0.1953, 0.0488, 0.0122, 0.00305,0.000762 nM antibody. In duplicate, 50 μL/well of the antibodies weretransferred into respective wells of the assay plate. To the antibodyalone plates, 50 μL/well of media was added to the inner 60 wells. Forthe cross-linker dilution series, goat anti-human IgG Fc specific(Jackson ImmunoResearch) was diluted to 800, 200, 50, 12.5, 3.125,0.7812, 0.1953, 0.0488, 0.0122, 0.00305 nM and 50 μL/well transferred tothe inner 60 wells to maintain a 4:1 ratio of anti-OX40 antibody andcross-linker. Growth media (150 μL) was added to the outer wells toprevent evaporation in the inner 60 wells. Plates were incubated at 37°C. for approximately 18 hours. Luciferase activity was quantified withBriteLite Plus (Perkin Elmer). Briefly, substrate was dissolved with 10mL of vendor-provided buffer and 75 μL substrate/well was added to theinner 60 wells of each plate. The plates were analyzed on the Victor5(Molecular Devices) using the Luminescence settings.

8.1.9. ADCC Reporter Assay

ADCC effector cells expressing human FcγRIII (Promega) were thawed andgrown as per protocol recommendations. Cells were split twice beforeuse. HEK293 cells stably transfected with either human or rhesus OX40were used as target cells. These cells were propagated in HyClone™ DMEMwith 10% heat inactivated FBS (Sigma) and 5 μg/mL Blasticidin (GibcoLife Technologies).

On the day prior to the assay, OX40-expressing HEK293 target cells wereharvested with 0.25% Trypsin (Gibco Life Technologies). Cells werewashed, counted, and plated at 10,000 cells/well in 96-well CostarPlates (Corning). Plates were incubated at 37° C. overnight in DMEM 10%FBS. ADCC Bioassay Effector Cells, Propagation Model protocol G7102 wasfollowed for the assay. Effector to Target cell ratio was 7.5:1.Luminescence was measured with EnSpire Alpha reader (Perkin Elmer) usingEnSpireManager software. Antibodies that were tested in this assayincluded isotype control antibody and anti-OX40 antibodies.

8.1.10. Anti-OX40 Antibody Binding to Activated Human CD4+ T Cells

Human PBMCs were isolated from buffy coats purchased from Stanford BloodCenter (Palo Alto, Calif.). Briefly, buffy coats were diluted in a 1:1ratio with PBS without magnesium and calcium (GE Healthcare). Dilutedblood (30 mL) was layered over 15 mL of 90% Ficoll-Paque Plus (GEHealthcare) prepared in PBS without magnesium and calcium (GEHealthcare) contained in SepMate tubes (Stemcell Technologies). Thetubes were spun at 1200 g for 10 minutes. The interphase was collectedand washed twice in 1×PBS. CD4+ T cells were isolated using StemcellTechnologies CD4 enrichment kit (Stem Cell Technologies). Cells wereresuspended to 2×10⁶ cells/mL in RPMI/10% FBS. Dynal CD3/28 beads (LifeTechnologies) were added at a 1:1 ratio. Cells were incubated on an endover end rotator at room temp for 20 minutes. The cells were cultured in6-well plates for 24 hours at 37° C.

After 24 hours, the beads were removed with a magnet. Cells were countedand resuspended to 1.5×10⁶/mL. An aliquot of the cell suspension (100μL) was used per stain. Test antibody was titrated in a 4-fold dilutionseries starting at 1 μg/mL. Cells were stained for 30 minutes and washedtwice. A 1:250 dilution of (4 μg/mL) of Goat anti Human Fcspecific-PE/well (Jackson ImmunoResearch) was added in 100 μL/well PBScontaining 1% BSA. Cells were stained for an additional 30 minutes andwashed twice, transferred to tubes and acquired using the BD LSRFortessa flow cytometer, and analyzed using FACSDiva analysis softwareversion 8.0.1.

8.1.11 Anti-OX40 Antibody Binding to Activated Cynomolgus T Cells

Cynomolgus monkey whole blood was purchased from Worldwide Primates. Forisolation of PBMCs, whole blood was diluted in a 1:1 ratio with PBSwithout magnesium and calcium (GE Healthcare). Diluted blood (30 mL) waslayered under 13 mL of 95% Ficoll-Paque Plus (GE Healthcare) prepared inPBS without magnesium and calcium (GE Healthcare) in 50-mL conicaltubes. The tubes were spun at 1000 g for 25 minutes. The interphase wascollected and washed twice in 1×PBS. Cells were resuspended to 2×10⁶cells/mL in RPMI/10% FBS. Cells were incubated for 72 hours with 10mg/mL phytohemagglutinin (PHA) (Sigma) and 100 U/mL recombinant humaninterleukin-2 (IL-2) (Proleukin®, Prometheus) in 6-well plates. After 24hours, cells were washed, counted and resuspended to 2×10⁶/mL. 100 μL ofthe cells were used per stain. Test anti-OX40 antibody was titrated in a4-fold dilution series starting at 1 μg/mL. Cells were stained for 30minutes and washed twice. A 1:250 dilution (4 μg/mL) of Goat anti-HumanIgG Fc specific-PE (Jackson ImmunoResearch) in 100 μL PBS containing 1%BSA was added per well. Cells were stained for an additional 30 minutesand washed twice, transferred to tubes and acquired using the BD LSRFortessa flow cytometer, and analyzed using FACSDiva analysis softwareversion 8.0.1.

8.1.12. Activated Human T Cell Proliferation and IFN-γ Induction

Human buffy coats were purchased from Stanford Blood Center (Palo Alto,Calif.). For isolation of human PBMCs, buffy coats were diluted in a 1:1ratio with PBS without magnesium and calcium (GE Healthcare). Dilutedblood (30 mL) was layered over 15 mL of 90% Ficoll-Paque Plus (GEHealthcare) prepared in PBS without magnesium and calcium (GEHealthcare) contained in SepMate tubes (Stemcell Technologies). Thetubes were spun at 1200 g for 10 minutes. The interphase was collectedand washed twice in 1×PBS. CD4+ T cells were isolated from the PBMCsusing EasySep CD4+ T cell enrichment kit (Stemcell Technologies). CD4+ Tcells were cultured at 2×10⁶ cells/mL in RPMI+10% FCS plus 2 μg/mL PHA(Sigma) and 20 IU/mL recombinant human IL-2 (Proleukin®, Prometheus) in6-well plates for 72 hours.

Biocoat T cell activation control plates-96-well plates (Corning) werecoated with 2 μg/mL goat anti-mouse IgG Fc-specific (JacksonImmunoResearch) and 2 μg/mL goat anti-human IgG-Fc specific (JacksonImmunoResearch) in 100 μL/well PBS overnight at 4° C. Plates wereblocked with 200 μL/well of 1% BSA (Rockland) in PBS for 30 minutes atroom temp. Plates were washed twice with 200 μL/well PBS. 4 ng/mL ofanti-human CD3 OKT3 (eBioscience) was added in 100 μL/well PBS andincubated for 90 minutes at 37° C. Plates were washed twice with 200μL/well PBS. A 3-fold dilution series of anti-OX40 antibody and isotypecontrol monoclonal antibody starting at 5 μg/mL was added to the platesin 100 μL PBS. The plates were incubated for 90 minutes at 37° C. Plateswere washed twice with 200 μL/well PBS. The washed PHA and IL-2activated CD4+ T cells (2×10⁵) were added to each well.

After 48 hours of culture at 37° C., 30 μL of supernatant from eachduplicate was pooled for IFN-γ analysis with Luminex (Millipore) andanalyzed on Bioplex Manager 6.0 (BioRad). Plates were pulsed with 0.25μCi ³H-thymidine (Perkin Elmer) overnight and harvested the followingmorning on Filtermats (Perkin Elmer) with 5 mL Ultima Gold Scintillationfluid (Perkin Elmer). Filtermats were counted on 1450 Microbeta WallacTrilux counter (PerkinElmer).

8.1.13. Human Regulatory T Cell Suppression Assays

Fresh peripheral blood mononuclear cells (PBMCs) were obtained fromAllCells or Stemcell Technologies. Cells were spun down, the cell pelletwas resuspended with 1×PBS and spun down once again at 1200 rpm for 10minutes at room temperature. Supernatants were removed and cells werethen resuspended with RoboSep buffer (Stemcell Technologies). Cellviability and cell count were determined using the Vi-Cell XR cellCounter Beckman Coulter. 100-150 million cells were set aside for CD4+ Tcell enrichment using Stemcell EasySep Human CD4+ T cell Enrichment Kit.The enriched CD4+ T cells were then depleted of CD25+ cells usingStemcell EasySep Human CD25+ Selection kit. This process resulted inpurified CD4+/CD25− responder T cells (Tresp). Residual PBMC were usedfor isolating regulatory T cells (Treg) following instructions from theStemcell EasySep Human CD4+/CD127low/CD49d− Regulatory T cell EnrichmentKit. After isolation of CD4+/CD25− Tresp and Treg, cells wereresuspended with RPMI 1640 with 10% heat inactivated FBS and 0.01 mM2-Mercaptoethanol at 1×10⁶ cells/mL and 5×10⁵ cells/mL respectively.

Treg Suppression assay was set up using two different ratios of Tresp toTreg at 2:1 and 4:1. For a 2:1 ratio, 5×10⁴ Tresp cells and 2.5×10⁴ Tregcells were added to 96-well U-bottom plates. For a 4:1 ratio, 5×10⁴Tresp cells and 1.25×10⁴ Treg cells were added to the 96-wells plate.Treg Suppression Inspector bead reagent (Miltenyi Biotec) was also addedto wells at 1:1 bead-to-cell ratio for stimulation. Anti-OX40 antibodyand isotype control human IgG₁ were tested in triplicate at 10 μg/mLfinal concentration in the absence or presence of F(ab′)₂ goatanti-human (GxHu) IgG, Fc specific (Jackson ImmunoResearch) at 1:4ratio. Plates were incubated at 37° C. in 5% CO₂ for four days. Plateswere treated with 1 μCi/well ³H-thymidine and further incubated foranother 16 hours at 37° C. in 5% CO₂. After incubation, plates wereharvested and proliferation measured using Ultima Gold Scintillationfluid (Perkin Elmer) and the 1450 Microbeta Wallac Trilux scintillationcounter (PerkinElmer).

8.1.14. Human Immune Cell-Engrafted PC-3 Mouse Tumor Model

On the day of inoculation, human T cells, autologous human moDC(monocyte-derived dendritic cells) and PC-3 cells were counted byVi-Cell XR (Beckman Coulter) and combined to deliver a subcutaneousinjection of 1×10⁷ PC-3, 1×10⁶ T cells and 5×10⁵ moDC per NSG mice(NOD.Cg-Prkdc^(scid) Il2rg^(tm1Wjl)/SzJ mouse) in 100 μL Dulbecco'sPhosphate Buffered Saline (DPBS) (GE Lifesciences). Treatment groups(n=8 mice/group) of 10 mg/kg isotype control monoclonal antibody and 10mg/kg Hu3738 were prepared in 200 μL DPBS for intraperitoneal injection.A single antibody dose was injected at the time of cell-mixtureinoculation. Measurement of tumor growth was assessed by standardcaliper measurement and tumor growth volume was calculated(Length×width×height/2).

8.1.15. Human PBMC GVHD Model in NSG Mice

Human peripheral blood mononuclear cells (PBMCs) were purchased fromAllCells (Oakland, Calif.). Immunodeficient NSG mice(NOD.Cg-Prkdc^(scid) Il2rg^(tm1Wjl)/SzJ) were inoculated with 2×10⁷human PBMC intraperitoneally on day 1. Anti-OX40 antibody Hu3738 orisotype control was administered intraperitoneally once a week startingon day 1. Once mice exhibited behavioral signs of graft-versus-hostdisease (GVHD) (e.g., hunched posture, ruffled fur), serum samples wereobtained, and levels of cytokines in the serum were determined using aLuminex bead array assay (Millipore).

Example 2: Generation and Humanization of Mouse Anti-OX40 Antibodies

Mice were immunized according to the methods known in the art (E.Harlow, D. Lane. Antibody: A Laboratory Manual, (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1998)). Isotype of eachmonoclonal antibody was determined using the Mouse Isotyping kit(Roche). Hybridoma clones producing antibodies of interest were purifiedand further characterized for affinity by surface plasmon resonance andfor ligand competition by FACS.

Cloning and construction of the expression vector were accomplished bymethods known in the art for expression of recombinant monoclonalantibodies.

Humanization of the antibody V region was carried out as outlined byQueen, C. et al. (Proc. Natl. Acad. Sci. USA, 1989; 86:10029-10033). Thecanonical structures of the CDRs were determined according to Huang etal. (Methods, 2005; 36:35-42). Human variable germline sequences withthe same or most similar CDR canonical structures were identified, andappropriate human V_(H), V_(L), and J segment sequences were selected toprovide the frameworks for the anti-OX40 variable region. At frameworkpositions in which the computer model suggested significant contact withthe CDRs, the amino acids from the murine anti-OX40 V regions weresubstituted for the original human framework amino acids(back-mutations).

Anti-OX40 mouse antibodies Mu3726, Mu3738, Mu3739, and Mu3741 werehumanized according to the method described above. The humanized versionof Mu3726 V_(H) was Hu3726 V_(H).1a which had human V_(H)4-28 frameworkregions, with seven back mutations of I48M, V67I, M69I, V71R, F78V,A93V, and R94K. Hu3726 V_(H).1a was combined with its respectivehumanized light chain Hu3726 V_(L).1b which had human VK1-39 frameworkregions, with two back mutations of Y48F and F71Y. The humanized versionof Mu3738 V_(H) was Hu3738 V_(H).1b which had human V_(H) 3-7 frameworkregions, with one back mutation of W47L. Hu3738 V_(H).1b was combinedwith its respective humanized light chain Hu3738 V_(L).1 which had humanVK4-1 framework regions and no back mutations. The humanized version ofMu3739 V_(H) was Hu3739 V_(H).1b which had human V_(H)1-69 frameworkregions, with four back mutations of M48I, V67A, E73T, and S76N. Hu3739V_(H).1b was combined with its respective humanized light chain Hu3739V_(L).1b which had human VK1-39 framework regions, with two backmutations of V58L and F71Y. The humanized version of Mu3741 V_(H) wasHu3741 V_(H).2b which had human V_(H)3-66 framework regions, with sevenback mutations of A24V, V48L, S49G, F67L, R71K, N76S, and L78V. Hu3741V_(H).2b was combined with its respective humanized light chain Hu3741V_(L).1c which had human VK1-39 framework regions, with two backmutations of Y36F and F71Y.

Example 3: Binding Affinity of the Anti-OX40 Antibodies

Table 3-1 below shows in vitro binding affinity data of exemplaryanti-OX40 antibody Hu3738, or literature anti-OX40 antibodies 11D4 or18D8 described in U.S. Pat. No. 7,960,515. Each of 11D4 and 18D8 is ahuman IgG₁, with a light kappa region.

As used herein, 11D4 has a V_(H) with amino acid sequence according to:

(SEQ ID NO: 61) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTIDYADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYC ARESGWYLFDYWGQGTLVTVSS,anda V_(L) with amino acid sequence according to:

(SEQ ID NO: 62) DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPP TFGGGTKVEIK.

18D8 has a V_(H) with amino acid sequence according to:

(SEQ ID NO: 63) EVQLVESGGGLVQPGRLSRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRENAKNSLYLQMNSLRAEDTALYYCAKDQSTADYYFYYGMDVWGQGTTVTVSS,anda V_(L) with amino acid sequence according to:

(SEQ ID NO: 64) EIVVTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPT FGQGTKVEIK.

Hu3738 exhibited potent binding properties to human OX40 by surfaceplasmon resonance, or in transfected Jurkat NF-κB reporter cellsexpressing human OX40 as measured in the assays of Example 1, and higherbinding affinity by SPR as compared with Hu3739 or Hu3741.

TABLE 3-1 Binding Properties of Exemplary Antibodies against Human OX40Jurkat cell surface Antibody K_(D) (M)* k_(d) (1/sec)* binding EC₅₀(ng/mL) 11D4 1.6E−09 2.7E−04 55 18D8 9.1E−09 1.1E−01 258 Hu3738 4.2E−084.2E−02 75 Hu3739 3.6E−07 1.6E−02 N/A Hu3741 3.0E−06 3.1E−01 N/A *asdetermined by surface plasmon resonance according to Example 1;exponential notation shown (e.g., 3.0E−09 = 3.0 × 10⁻⁹); N/A = notavailable.

Exemplary anti-OX40 antibody Hu3738 exhibited cross reactivity againstcynomolgus or rhesus monkey OX40, but did not demonstrate significantbinding against mouse or rat OX40. The binding activity of Hu3738 torecombinant human or cynomolgus (cyno) OX40, or to cell-surface human orrhesus monkey OX40, as determined by the assays described in Example 1is summarized in Table 3-2.

TABLE 3-2 Binding Properties of Hu3738 against Human, Cynomolgus orRhesus OX40 Assay EC₅₀ (nM) ELISA Human 0.044 Cyno 0.039 Jurkat Human0.50 NF-κB cell rhesus 2.1

Example 4: In Vitro Biological Activity of Anti-OX40 Antibody Hu3738

To assess binding of Hu3738 to endogenously expressed human OX40, bothactivated CD4+ T cells and unstimulated peripheral blood mononuclearcells (PBMC) were examined by flow cytometry. Table 4-1 summarizes thedata for binding of Hu3738 on cell surface OX40 on CD3/CD28 beadactivated human CD4+ T cells, or phytohemagglutinin (PHA) andinterleukin-2 (IL-2) activated cynomolgus monkey CD4+ T cells, accordingto the assays described in Example 1. As shown in Table 4-1, Hu3738potently bound to OX40 on activated CD4+ T cells in human and cynomolgusT cell cultures.

TABLE 4-1 CD4+ T cell Binding of Exemplary Antibody Hu3738 OX40 SpeciesEC₅₀ (nM) Human 0.053 Cynomolgus 0.024

The subnanomolar binding of human OX40 by Hu3738 afforded functionalactivation as demonstrated by increased proliferation of humanperipheral blood CD4+ T cells and enhanced interferon-γ production byhuman CD4+ T cells after in vitro treatment with Hu3738 according to theassays described in Section 8.1.12 (FIGS. 1A, 1B). As shown in a typicalexperiment depicted in FIG. 1A, Hu3738 effected an increasedproliferation of human peripheral blood CD4+ T cells of from about 1.5-to about 6-fold as compared to isotype control huIgG₁, which wascomparable or greater than the increase in proliferation observed when Tcells were dosed with an equivalent amount of literature anti-OX40antibody 11D4 or 18D8. Hu3738 showed EC₅₀=0.11 nM (16 ng/mL) in anaverage of runs from eight donors.

FIG. 1C shows that proliferation of human peripheral blood CD4+ T cellsafter in vitro treatment with Hu3738 was similar to literature anti-OX40antibody 1A7 over a broad range of antibody concentrations from about0.001 to about 1 μg/mL, when each was tested according to the T cellproliferation assay described in Section 8.1.12.

Antibody 1A7 described in US publication no. 2015/0307617 is a humanIgG₁ with kappa light chains, having a V_(H) amino acid sequenceaccording to:

(SEQ ID NO: 69) EVQLQQSGPELVKPGASVKISCKASGYTFTDSYMSWVKQSHGKTLEWIGDMYPDNGDSSYNQKFREKVTLTVDKSSTTAYMEFRSLTSEDSAVYYC VLAPRWYFSVWGTGTTVTVSS,anda V_(L) amino acid sequence according to:

(SEQ ID NO: 70) DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLRSGVPSRFSGSGSGKDYFLTISNLEQEDVAAYFCQQGHTLPP TFGGGTKLEIK.

As shown in a typical experiment depicted in FIG. 1B, IFN-γ productionincreased in human CD4+ T cells from about 2- to about 10-fold whentreated with Hu3738 across the concentration range tested. With regardto IFN-γ production, Hu3738 exhibited EC₅₀=0.16 nM (24 ng/mL) in anaverage of runs from nine donors. Literature anti-OX40 antibodies 11D4and 18D8 also demonstrated a similar effect on IFN-γ production underthese assay conditions.

FIG. 1D shows that Hu3738 effects a higher IFN-γ production increase inhuman CD4+ T cells as compared with literature anti-OX40 antibody 1A7,when each was tested according to the T cell IFN-γ production assaydescribed in Section 8.1.12.

In addition to the downstream signaling activation effects in increasingproliferation of CD4+ T cells and enhancing production of IFN-γ, theexemplary anti-OX40 antibody Hu3738 also inhibited human T regulatorycell activity in vitro, suggesting that T regulatory cells within asolid tumor, which can inhibit an immunological response by the body toattack the tumor, may be suppressed with administration of Hu3738.

The effect of Hu3738 on T regulatory activity was assessed in vitroaccording to the assay described in Section 8.1.13. AutologousCD4+/CD25− T responder (Tresp) cells were co-cultured withCD4+/CD25+/CD127low T regulatory (Treg) cells and activator beads (Insp)at a 2:1 or a 4:1 Tresp:Treg ratio (FIGS. 2A, 2B). In the absence ofTreg, the Tresp cells proliferated in response to the activator beads.In the presence of Treg, proliferation was inhibited. Inclusion of 10μg/mL Hu3738 in the culture media had no impact on the Treg mediatedsuppression. The isotype control used for these T regulatory suppressionassay was huIgG₁ with the constant region variants L234A and L235A.Separate experiments performed using the huIgG₁ isotype control withcross-linker showed no effect on the assay.

By contrast, in the presence of an exogenous cross-linker, Hu3738resulted in complete restoration of the Tresp proliferation (FIGS. 2Aand 2B). Hu3738 in the presence of cross-linker enhanced proliferationin this assay above the level of the Tresp response to the activatorbeads alone. This result suggested that OX40 signals may overcome Tregulatory cell mediated suppression and may enhance antigen-specificresponses consistent with results reported above.

ADCC activity mediated by Hu3738 was evaluated using a commerciallyavailable ADCC reporter assay as described in Section 8.1.9. This assayutilized engineered Jurkat cells expressing human FcγRIIIa and a nuclearfactor of activated T cells (NFAT) reporter as the effector cells. HEK293 cells expressing human OX40 were used as target cells, and theanti-OX40 antibody was expected to bind to OX40 expressed on the targetcells. Additionally, the Fc region of Hu3738 was expected to bind toFcγRIIIa receptors on the cell surface of the reporter cells. Thesebinding events would have resulted in multiple cross-linking of the twocell types leading to ADCC reporter activity activation, an effect thatwas measured through luminescence readout as a result of the NFATpathway activation. Compared to isotype control, Hu3738 increased ADCCreporter activity, with an EC_(50=0.51) nM (77 ng/mL).

Example 5: Epitope Classification of Exemplary Anti-OX40 Antibodies

8.5.1. Binding of Hu3738 with Human/Mouse Chimeric OX40

Soluble OX40L blocked Hu3738 binding to OX40 with an IC₅₀=67 pM (10ng/mL) in a human OX40-expressing Jurkat cell assay described in Section8.1.5 (FIG. 3), suggesting that Hu3738 bound to human OX40 in the ligandbinding region of the molecule.

For more detailed epitope mapping of Hu3738 antibody binding, a seriesof cell lines expressing human/mouse cysteine-rich domain (CRD)-swappedOX40 molecules were created. This method is based on the observationthat Hu3738 does not bind to mouse OX40 (FIG. 4B). A sequence alignmentof human OX40 (SEQ ID NO:1) with mouse OX40 (SEQ ID NO:3) is shown inFIG. 4A. From the analysis of the sequences, a series of 293stransfectants expressing chimeric versions of the human OX40 receptorwith swapped-in mouse CRD sequences were generated and stained withHu3738.

The amino acid sequences (including signal sequences) of the human-mouseOX40 receptor chimeras, with murine swapped-in regions indicated asunderline, are as follows. The human OX40 chimera with murine CRDIreplacing human CRDI has an amino acid sequence according to:

(SEQ ID NO: 5) MCVGARRLGRGPCAALLLLGLGLSTVTGLNCVKHTYPSGHKCCRECQPGHGMVSRCDHTRDTLCHPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLGLVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI,the human OX40 chimera with murine CRDII replacing human CRDII has anamino acid sequence according to:

(SEQ ID NO: 6) MCVGARRLGRGPCAALLLLGLGLSTVTGLHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCETGFYNEAVNYDTCKQCTQCNHRSGSELKQNCTPTQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLGLVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI,the human OX40 chimera with murine CRDIII replacing human CRDIII has anamino acid sequence according to:

(SEQ ID NO: 7) MCVGARRLGRGPCAALLLLGLGLSTVTGLHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRPGTQPRQDSGYKLGVDCVPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLGLVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI,the human OX40 chimera with murine CRDIV replacing human CRDIV has anamino acid sequence according to:

(SEQ ID NO: 8) MCVGARRLGRGPCAALLLLGLGLSTVTGLHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGNNQACKPWTNCTLSGKQTRHPASDSLDAVCEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLGLVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI,and the human OX40 chimera with murine CRDII and CRDIII replacing humanCRDII and CRDIII has an amino acid sequence according to:

(SEQ ID NO: 9) MCVGARRLGRGPCAALLLLGLGLSTVTGLHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCETGFYNEAVNYDTCKQCTQCNHRSGSELKQNCTPTQDTVCRCRPGTQPRQDSGYKLGVDCVPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLGLVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI.

Binding determination of Hu3738 to this series of chimeras was performedaccording to the assay described in Section 8.1.7 to localize thebinding site to specific CRD regions. A loss in binding suggested whichCRDs were critical for OX40 recognition by a particular antibody. Inthis instance, the absence of detectable binding to a specificCRD-swapped region in the mouse suggested the region of human OX40receptor recognized by Hu3738. The anti-OX40 antibody Hu3738 was shownto lose binding when the human CRDII was replaced with the correspondingmouse CRDII, consistent with Hu3738 binding to CRDII of human OX40 (FIG.4B).

8.5.2. Competition Assay with Exemplary Anti-OX40 Antibody Hu3738 Boundto Human OX40

Additional literature humanized anti-OX40 antibodies were generated tocompare with the exemplary anti-OX40 antibodies of the disclosure.Antibodies 106-222 and 119-122, described in US publication no.2013/0280275, are human IgG₁ with kappa light chains.

Antibody 106-222 has a V_(H) amino acid sequence according to:

(SEQ ID NO: 65) QVQLVQSGSELKKPGASVKVSCKASGYTFTDYSMHWVRQAPGQGLKWMGWINTETGEPTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCANPYYDYVSYYAMDYWGQGTTVTVSS,anda V_(L) amino acid sequence according to:

(SEQ ID NO: 66) DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKWYSASYLYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYSTPRTF GQGTKLEIK.

Antibody 119-122 has a V_(H) amino acid sequence according to:

(SEQ ID NO: 67) EVQLVESGGGLVQPGGSLRLSCAASEYEFPSHDMSWVRQAPGKGLELVAAINSDGGSTYYPDTMERRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHYDDYYAWFAYWGQGTMVTVSS,anda V_(L) amino acid sequence according to:

(SEQ ID NO: 68) EIVLTQSPATLSLSPGERATLSCRASKSVSTSGYSYMHWYQQKPGQAPRLLIYLASNLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSR ELPLTFGGGTKVEIK.

Binding of Hu3738 to cell-surface expressed OX40 was shown to becompetitive with some but not all literature anti-OX40 antibodies bydirect competition studies. As shown in FIG. 5, Jurkat-NF-κB-huOX40cells treated with a dose titration of literature antibodies, then weresubsequently subjected to 2 μg/mL of fluorescent ALEXA FLUOR®647-labeled Hu3738 to determine binding competition. Analysis wasperformed by flow cytometry. Literature anti-OX40 antibodies 106-222 or1A7 were competitive with Hu3738. However, antibodies 11D4, 18D8, or119-122 did not compete with Hu3738 up to 100 μg/mL.

Example 6: Ox40 Activation by Exemplary Anti-OX40 Antibody Hu3738

The Jurkat NF-κB cell data highlights the activating ability ofexemplary anti-OX40 antibody Hu3738, even in the absence of across-linker (FIGS. 6A, 6B). As shown in FIG. 6A, the only anti-OX40antibodies that demonstrated significant NF-κB signaling activity acrossthe range of concentrations from about 0.001 to about 100 μg/mL antibodywere Hu3738 and the corresponding murine Mu3738. Literature anti-OX40antibodies 11D4, 18D8, 106-222, and 119-122 each lacked a significantNF-κB signaling effect up to about 100 μg/mL antibody.

The activity of Hu3738 in the absence of exogenous cross-linkingcontrasted to the lack of activity of other literature anti-OX40antibodies under the same assay conditions. A summary of the NF-κB cellsignaling data is shown in Tables 6-1 and 6-2. Notably, though Hu3738competed to bind human OX40 with literature anti-OX40 antibodies 106-222and 1A7, Hu3738 exhibited a different functional activity compared toeach of the antibodies in the absence of a cross-linker.

TABLE 6-1 NF-κB signaling in Jurkat-NF-κB-huOX40 cells EC₅₀ Without EC₅₀With Antibody Cross-linker (nM) Cross-linker (nM) Hu3738 20 0.088 11D4NS* 0.94 18D8 NS* 0.50 106-222 NS* 0.63 119-122 NS* 0.34 Isotype NS* NS**NS = no significant NF-κB signaling up to 100 μg/mL antibody; N/A = notavailable.

In addition to its ability to effect NF-κB signaling in the absence ofexogenous cross-linker in the Jurkat-NF-κB-huOX40 cell, Hu3738 alsodemonstrated greater potency as measured by EC₅₀ with cross-linker,compared with literature anti-OX40 antibodies 11D4, 18D8, 106-222, and119-122 (Table 6-1).

A side-by-side comparison of Hu3738 with 1A7 is shown in FIG. 6B andsummarized in Table 6-2 below. In addition to the lack of NF-κBsignaling in the absence of exogenous cross-linker in theJurkat-NF-κB-huOX40 cell, each of the literature anti-OX40 antibodiesdescribed above also exhibited a lower EC₅₀ as compared with Hu3738.

TABLE 6-2 NF-κB signaling in Jurkat-NF-κB-huOX40 cells EC₅₀ Without EC₅₀With Antibody Cross-linker (nM) Cross-linker (nM) Hu3738 22 0.020 1A7NS* 0.066 Isotype NS* NS* *NS = no significant NF-κB signaling up to 100μg/mL antibody.

Example 7: Anti-Tumor Activity of Hu3738 in Human Cell Adoptive TransferModel in Mouse

Hu3738 demonstrated anti-tumor activity in an in vivo NSG mouse modelafter a single dose inoculation with human PC3 cells, T cells andautologous monocyte-derived dendritic cells (moDC) according to theprotocol described in Section 8.1.14 (FIG. 7). On the day ofinoculation, human T cells, moDC and PC3 cells were delivered bysubcutaneous injection to each NSG mouse. Isotype control monoclonalantibody or Hu3738 (10 mg/kg) was each dosed intraperitoneally peranimal in each treatment group (n=8), with the antibody dose injected atthe time of inoculation. Measurement of tumor growth was assessed bystandard caliper measurement and tumor growth volume was calculated(Length×width×height/2).

As shown in FIG. 7, Hu3738 significantly inhibited the growth of the PC3tumor in NSG mice 17 days post-inoculation as compared with anequivalent dose of isotype control antibody.

Example 8: In Vivo Immune Activation in Human PBMC GVHD Model

NSG mice were inoculated with human PBMC intraperitoneally. The micewere then treated with 1 mg/kg Hu3738 or huIgG₁ isotype control q7d×4(i.e., once every 7 days for a total of 4 doses), with the first dose atday 1 immediately after inoculation with human cells. On day 22, themice were sacrificed and levels of cytokines in the serum weredetermined using a Luminex bead array assay (Millipore).

The results in FIG. 8 demonstrated that enhancement in interleukin-8(IL-8), granulocyte macrophage colony-stimulating factor (GM-CSF), tumornecrosis factor alpha (TNF-α), and interferon-gamma (IFN-γ) was observedafter dosing of anti-OX40 antibody Hu3738 as compared with isotype,suggestive of an increase in the immunological response in the mouse dueto Hu3738.

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

1. An anti-OX40 antibody which is an IgG antibody consisting of heavychains each consisting of the amino acid sequence of SEQ ID NO:41 andlight chains each consisting of the amino acid sequence of SEQ ID NO:51.2.-20. (canceled)
 21. A pharmaceutical composition comprising theanti-OX40 antibody of claim 1, and a pharmaceutically acceptablecarrier.
 22. An anti-OX40 antibody which is an IgG antibody consistingof heavy chains each consisting of the amino acid sequence of SEQ IDNO:42 and light chains each consisting of the amino acid sequence of SEQID NO:51.
 23. A pharmaceutical composition comprising the anti-OX40antibody of claim 1, and a pharmaceutically acceptable carrier.